DNA sequence encoding enzymes of clavulanic acid biosynthesis

ABSTRACT

DNA sequences are provided which encode the enzymes required for clavulanic acid synthesis. A process is provided for producing clavulanic acid in a transformant of a non-clavulanate-producing host.

[0001] This invention is a continuation-in-part of application Ser. No. 08/790,462, filed Jan. 29, 1997, which is a continuation-in-part of application Ser. No. 08/567,801, filed Dec. 6, 1995, which is a continuation of application Ser. No. 08/134,018, filed Oct. 8, 1993, now abandonded.

[0002] This invention relates to methods for the production of the antibiotic, clavulanic acid.

BACKGROUND OF THE INVENTION

[0003] Clavulanic acid is a broad spectrum beta-lactamase inhibitor and is an important antibiotic for the treatment of infectious diseases. It is produced commercially by the gram-positive mycelial prokaryote Streptomyces clavuligerus, which also produces the β-lactam antibiotics penicillin N, desacetoxy cephalosphorin C and cephamycin C. Until recently, however, the pathway employed for clavulanic acid biosynthesis was much less well understood than the pathways leading to these other antibiotics.

[0004] Without knowledge of the pathway for clavulanic acid biosynthesis, it was not possible to isolate the genes coding for the key enzymes and to manipulate these genes to increase antibiotic yield or permit production of the antibiotic in heterologous systems.

[0005] One of the earliest enzymes of the pathway to be purified and characterised was clavaminic acid synthase. Two isozymes have now been identified and characterised (Marsh et al., (1992), Biochem., vol. 31, pp. 12648-657).

[0006] European Patent Application 0349121 describes a 6.8 kb Bgl II restriction fragment isolated from S. clavuligerus that encodes a portion of the genetic information involved in clavulanic acid synthesis. No further characterization of this fragment was performed nor was the utility of this fragment determined.

[0007] Marsh et al (1992, Biochem. vol 31, pp. 12648-657) cloned and sequenced two isozymes of clavaminate synthase cs1 and cs2 separated by 28 kb, however their location relative to the cephamycin and penicillin biosynthetic clusters was not determined. Ward and Hodgson (1993, FEMS Microbiol. Lett. vol 110, pp. 239-242) reported on the occurrence of a biosynthetic gene cluster for clavulanic acid. Aidoo et al (1994, Gene vol 147, pp. 41-46) cloned and sequenced cla the gene encoding a protein (possibly proclavaminic acid amidinohydrolase) involved in clavulanic acid production. They reported that this gene was upstream from and adjacent to an ORF with the identical sequence of cs2. Paradkar and Jensen (1995, J Bacteriol, vol 177, pp. 1307-1314) further analyzed a 6.6 kb Bgl II fragment from S. clavuligerus comprising cs2 and through gene disruption experiments demonstrated its role in clavulanic acid synthesis.

[0008] Until the work of the present inventors, the complete complement of genes required for clavulanic acid synthesis had not been identified.

[0009] The present inventors have now isolated, cloned and sequenced a 15 kb DNA fragment from S. clavuligerus which encodes 10 ORFs. Within this 15 kb of DNA lies an 11.6 kb Eco RI fragment which codes for eight proteins and enables the production of clavulanic acid by transformants of non-clavulanic-producing organisms. This 11.6 kb fragment includes 8 complete ORFs (ORFs 2 to 9), two of which have been previously characterized (Marsh et al 1992, Paradkar and Jensen 1995). ORF1, which is incomplete, is not involved in clavulanic acid synthesis, ORF4 encodes the CLA protein and ORF5 is cs2, encoding one of the isozymes of clavaminate synthase. The function of the other remaining ORFs within this 11.6 kb fragment or their role in clavulanic acid synthesis is unknown.

SUMMARY OF THE INVENTION

[0010] An isolated genomic DNA molecule of 15 kb is provided comprising the nucleotide sequence set out in SEQ ID NO:1. This DNA molecule comprises 10 ORFs, eight of which are involved in clavulanic acid synthesis. A process is provided for producing clavulanic acid in a transformant of a non-clavulanate-producing host.

[0011] The present invention provides isolated DNA molecules comprising the nucleotide sequence of SEQ ID NO: 15, 16, and 19 to 21.

[0012] Furthermore, the present invention is directed to DNA molecules comprising the nucleotide sequences that encode the amino acid sequence found in SEQ ID NO's: 4, 5, and 8 to 11.

[0013] This invention also embraces DNA molecules comprising a nucleotide sequence of ORFs 2, 3, 6, 7, 8 and 9 and their associated non-transcribed regions. Such isolated DNA molecules have the nucleotide sequences found in SEQ ID NO's: 15, 16 and 19 to 21.

[0014] This invention also embraces DNA molecules comprising the nucleotide sequences encoding the amino acid sequences of SEQ ID NO':s 4, 5, and 8 to 11. These amino acid sequences correspond to the expression products of ORFs 2, 3, 6, 7, 8 and 9 as defined in SEQ ID NO's: 4, 5 and 8 to 11.

[0015] This invention is also directed to recombinant vectors that comprise DNA molecules as defined above where hosts that have been transformed with these recombinant vectors.

[0016] This invention is also directed to the process for producing clavulanic acid in a non-clavulantic acid producing host that comprises transforming the host with a DNA molecule as defined above and culturing the host under suitable conditions to produce clavulanic acid.

[0017] Furthermore, this invention is directed to processes for enhancing clavulanic acid production in a clavulanic acid producing host comprising transforming the host with a DNA molecule as defined above.

DESCRIPTION OF DRAWINGS

[0018] The invention, as exemplified by a preferred embodiment, is described with reference to the accompanying drawings in which:

[0019]FIG. 1 shows the N terminal amino acid sequence of CLA (amino acid residues 1-25 of SEQ ID NO:6), the potential codon corresponding with this sequence (SEQ ID NO:24) and the nucleotide sequence of a probe (SEQ ID NO:25) directed to the underlined region of the sequence. The nucleotide sequence of the actual sequence is defined in SEQ ID NO:1, specifically nucleotides 5687-5710 of SEQ ID NO:1, 3665-3678 of SEQ ID NO:13, or 34-57 of SEQ ID NO:17.

[0020]FIG. 2 shows the location of the open reading frames downstream from pcbC.

[0021]FIG. 3 shows a partial restriction map in the region surrounding cla (ORF4), SEQ ID NO:6.

[0022]FIG. 4 shows a shuttle vector used for disruption of the cla gene.

[0023]FIG. 5 shows a photograph of an agar plate bearing cultures of S. lividans transformants.

[0024]FIG. 6 shows a Southern blot of NcoI digests of genomic DNA from five presumptive mutants (lanes 1-5) and from wild-type S. clavuligerus (lane 6). Panel A: membranes probed with cla-specific probe. Panel B: membranes probed with tsr-specific probe.

[0025]FIG. 7 shows restriction enzyme maps of S. clavuligerus DNA inserts in cosmids. FIG. 7(A). Restriction enzyme map of cosmid K6L2. FIG. 7 (B). Partial restriction enzyme map of cosmid K8L2. FIG. 7 (C). Restriction map of cosmids K6L2 and K8L2 indicating location of pcbC gene in relation to cla. FIG. 7(D). The 2.0 kb NcoI fragment encompassing the cla gene used in generating nested deletions for sequencing. Abbreviations: Ba, BamHI; B,BglII; E,EcoRI; K,KpnI; N, NcoI; S,SalI; and Sm,SmaI.

[0026]FIG. 8 shows the deduced amino acid sequence (SEQ ID. NO:3) of ORF1.

[0027]FIG. 9 shows the deduced amino acid sequence (SEQ ID NO:4) of ORF2.

[0028]FIG. 10 shows the deduced amino acid sequence (SEQ ID NO:5) of ORF3.

[0029]FIG. 11 shows the deduced amino acid sequence (SEQ ID NO:6) of ORF4.

[0030]FIG. 12 shows the deduced amino acid sequence (SEQ ID NO:7) of ORF5.

[0031]FIG. 13 shows the deduced amino acid sequence (SEQ ID NO:8) of ORF6.

[0032]FIG. 14 shows the deduced amino acid sequence (SEQ ID NO:9) of ORF7.

[0033]FIG. 15 shows the deduced amino acid sequence (SEQ ID NO:10) of ORF8.

[0034]FIG. 16 shows the deduced amino acid sequence (SEQ ID NO:11) of ORF9.

[0035]FIG. 17 shows the deduced amino acid sequence (SEQ ID NO:12) of ORF10.

[0036] FIGS. 18 to 23. The upper panel shows the results of Southern hybridizations using the probes and gene fragments as set forth in the restriction maps of the lower panel.

[0037]FIG. 18 shows the construction of the orf-2 mutant.

[0038]FIG. 19 shows the construction of the orf-3 mutant.

[0039]FIG. 20 shows the construction of the orf-6 mutant.

[0040]FIG. 21 shows the construction of the orf-7 mutant.

[0041]FIG. 22 shows the construction of the orf-8 mutant.

[0042]FIG. 23 shows the construction of the orf-9 mutant.

[0043]FIG. 24, high pressure liquid chromatography (HPLC) analysis of clavulanic acid in culture supernatants. Culture supernatants from 96 hour Starch- Asparagine medium-grown cultures of either wild type S. clavuligerus, or one of the gene disruption mutants were analyzed by HPLC. FIGS. 24 (A) and (B) show elution profiles of culture supernatants obtained from wild type (FIG. 24 (A) or ORF7 gene disruption mutant (FIG. 24 (B)). During this analysis, Clavulanic acid eluted with a retention time of approximately 10.13 min. FIGS. 24 (C), (D) and (E), show elution profiles of clavulanic acid standard (FIG. 24 (C)) and culture supernatants obtained from wild type (FIG. 24 (D)) and ORF8 disruption mutant (FIG. 24 (E)). During this analysis, clavulanic acid eluted with a retention time of approximately 9.1 min. Differences in retention times are due to the use of different columns between analysis of the samples. Similar HPLC profiles were seen for all of the other disruption mutants, including ORF2, ORF3, ORF6 or ORF9, and indicate that none of the mutants produced clavulanic acid under these culture conditions.

DETAILED DESCRIPTION OF THE INVENTION

[0044] Production of penicillin and cephamycin antibiotics in S. clavuligerus starts with the conversion of lysine to α-aminoadipic acid (Madduri et al., (1989), J. Bacteriol., v. 171, pp. 299-302; (1991), J. Bacteriol., v. 173, pp. 985-988). α-Aminoadipic acid then condenses with cysteine and valine to give δ-(L-α-aminoadipyl)-L cysteinyl-D-valine (ACV) by the action of aminoadipyl cysteinyl-valine synthetase (ACVS). ACV is converted by isopenicillin N synthase (IPNS) to isopenicillin N, and, through a series of reactions, to desacetoxycephalosporin C and ultimately to cephamycin C (Jensen et al., (1984), Appl. Microbiol. Biotechnol., v. 20, pp 155-160).

[0045] The ACVS of S. clavuligerus has been purified and partially characterized by three separate groups, and estimates of its molecular weight vary from 350,000 to 500,000 Da (Jensen et al., (1990) J. Bacteriol., v. 172, pp. 7269-7271; Schwecke et al., (1992), Eur. J. Biochem., v. 205, pp. 687-694; Zhang and Demain, (1990), Biotech Lett., v. 12, pp. 649-654). During their purification, Jensen et al. observed a 32,000 Da protein which co-purified with ACVS despite procedures which should remove small molecular weight components. It has now been found that this protein is not related to ACVS but rather to clavulanic acid biosynthesis. It has been designated CLA.

[0046] In accordance with one embodiment of the invention, the present inventors have identified, cloned and sequenced the gene (cla) encoding this protein.

[0047] In accordance with a further embodiment of the invention, the inventors have cloned and sequenced a 15 kb stretch of genomic DNA from S. clavuligerus which includes the cla gene. Within this 15 kb sequence, the inventors have identified an 11.6 kb DNA fragment which, when introduced into the non-clavulanate producer S. lividans as described in Example 4, enabled that species to produce clavulanic acid. This indicates that the 11.6 kb fragment contains all the genetic information required for clavulanate production.

[0048] As will be understood by those skilled in the art, the identification of the DNA sequence encoding the enzymes required for clavulanate synthesis will permit genetic manipulations to modify or enhance clavulanate production. For example, clavulanate production by S. clavuligerus may be modified by introduction of extra copies of the gene or genes for rate limiting enzymes or by alteration of the regulatory components controlling expression of the genes for the clavulanate pathway.

[0049] Heterologous organisms which do not normally produce clavulanate may also be enabled to produce clavulanate by introduction, for example, of the 11.6 kb DNA sequence of the invention by techniques which are well known in the art, as exemplified herein by the production of S. lividans strains capable of clavulanate synthesis. Such heterologous production of clavulanic acid provides a means of producing clavulanic acid free of other contaminating clavams which are produced by S. clavuligerus.

[0050] Suitable vectors and hosts will be known to those skilled in the art; suitable vectors include pIJ702, pJOE829 and pIJ922 and suitable hosts include S. lividans, S. parvulus, S. griseofulvus, S. antibioticus and S. lipmanii.

[0051] Additionally, the DNA sequences of the invention enable the production of one or more of the enzymes of the clavulanate pathway by expression of the relevant gene or genes in a heterologous expression system.

[0052] The DNA sequences coding for one or more of the pathway enzymes may be introduced into suitable vectors and hosts by conventional techniques known to those skilled in the art. Suitable vectors include pUC118/119 and pET-11 and suitable hosts include many organisms, including E. coli strains such as MV1193 and BL21(DE3).

[0053] An oligonucleotide probe (SEQ ID NO:25) based on the N-terminal amino acid sequence of CLA (amino acid residues 1-25 of SEQ ID NO:6) was constructed as shown in FIG. 1 and was used to isolate the gene coding for the protein from S. clavuligerus, as described in Example 1.

[0054] The gene was found to be located in the S. clavuligerus chromosome about 5.7 kb downstream of pcbC, the gene which encodes isopenicillin N synthase. The gene contains a 933 bp open reading frame (ORF), encoding a protein of molecular weight 33,368. The deduced amino acid sequence was compared to database sequences and showed greatest similarity to enzymes associated with arginine metabolism, notably agmatine, ureohydrolase and arginases.

[0055] When an internal fragment of the cla gene was labelled and used to probe restriction endonuclease digests of genomic DNA from a variety of other Streptomyces and related species, evidence of homologous sequences was seen only in other clavulanic acid or clavam metabolite producers, including Streptomyces jumonjinensis, Streptomyces lipmanii and Streptomyces antibioticus. No cross reactivity was seen to the 13-lactam producing species Nocardia lactamdurans, Streptomyces griseus or Streptomyces cattleya, nor to any of a variety of other Streptomyces species which do not produce β-lactam compounds, including S. fradiae ATCC 19609, S. venezuelae 13s and S. griseofulvus NRRL B-5429.

[0056] Disruption of the cla gene, as described in Example 3, led to loss of the ability to synthesise clavulanic acid.

[0057] A 15 kb DNA sequence extending downstream from pcbC was cloned and sequenced as described in Example 5. The nucleotide sequence is shown in SEQ ID NO:1. When this (SEQ ID NO:1) sequence information was analysed for percent G+C as a function of codon position (Bibb et al., (1984), Gene, v. 30, pp. 157-166), ten complete ORFs were evident, as shown in FIG. 2. ORF 4 corresponds to cla. ORF 1,7 & 8 are oriented in the opposite direction to pcbC. ORFs 2-6 and ORF 10 are all oriented in the same direction as pcbC. ORFs 2 and 3, and ORFs 4 and 5 are separated by very short intergenic regions suggesting the possibility of transcriptional and translational coupling. Table 1 summarises the nucleotide sequences and lengths of ORFs 1-10. TABLE 1 Start location End location Length Size of ORF SEQ ID ORF# (bp) (bp) (bp) (aa residues) NO.  1* 1764 109 1656 552 14 2 2257 3978 1722 574 15 3 3981 5522 1542 514 16 4 5695 6636 942 314 17 5 6652 7629 978 326 18 6 7936 9117 1182 394 19 7 9282 10949 1668 556 20  8* 12337 11039 1299 433 21  9* 13406 12663 744 248 22 10  13810 15036 1227 409 23

[0058] When the predicted amino acid sequences of proteins encoded by ORFs 1-10 were compared to protein sequence databases, some similarities were noted in addition to the already mentioned similarity between CLA and enzymes of arginine metabolism. ORF 1 (SEQ ID NO's: 3 and 14) showed a low level of similarity to penicillin binding proteins from several different microorganisms which are notable for their resistance to β-lactam compounds.

[0059] An EcoRI fragment of the 15 kb DNA sequence, containing 11.6 kb DNA, was cloned into a high copy number shuttle vector and introduced into S. lividans, as described in Example 4. Of seventeen transformants examined, two were able to produce clavulanic acid, indicating that the 11.6 kb fragment contains all the necessary genetic information for clavulanic acid production.

[0060] This 11.6 kb fragment encompasses ORF 2 to ORF 9 of the 15 kb DNA sequence.

[0061] ORF 2 (SEQ ID NO's:4 and 15) shows a high degree of similarity to acetohydroxyacid synthase (AHAS) enzymes from various sources. AHAS catalyses an essential step in the biosynthesis of branched chain amino acids. Since valine is a precursor of penicillin and cephamycin antibiotics, and valine production is often subject to feedback regulation, it is possible that a deregulated form of AHAS is produced to provide valine during the antibiotic production phase. Alternatively, an AHAS-like activity may be involved in clavulanic acid production. While the presently recognized intermediates in the clavulanic acid biosynthetic pathway do not indicate a role for AHAS, the final step in the biosynthetic pathway, conversion of clavaminic acid to clavulanic acid, requires NADPH, and either pyruvate or α-ketobutyrate as well as other cofactors (Elson et al., (1987), J. Chem. Soc. Chem. Commun., pp. 1739-1740). It is striking that these same substrates and cofactors are required for AHAS activity. Perhaps the conversion of clavaminate to clavulanate actually involves several steps, one of which is catalyzed by an AHAS-like activity. ORFs 3 (SEQ ID NO's:5 and 16) does not show a significant similarity to any proteins in the data bases. ORF 6 (SEQ ID NO's:8 and 19) shows similarity to ornithine acetyltransferase. Ornithine has been suggested to be the immediate precursor of a 5-C fragment of the clavulanic acid skeleton, but the details of the reaction required for the incorporation of ornithine are unknown. ORF 7 (SEQ ID NO's:9 and 20) shows weak similarity to protein XP55 from S. lividans, and a lower level of similarity to oligopeptide binding proteins from various other species. Similarly, ORF 8 (SEQ ID NO:10 and 21) shows weak similarity to several transcription activator proteins, and ORF 9 (SEQ ID NO's: 11 and 22) shows weak similarity to ribitol 5 P0₄ dehydrogenase-type enzymes. ORF 10 (SEQ ID NO:12 and 23) shows a high similarity to cytochrome P450 type enzymes from other Strepomyces species.

[0062] ORF5 has now been identified as the gene for clavaminate synthase II (Marsh (1993) supra).

[0063] When a plasmid isolated from one of the two clavulanic acid-producing transformants was retransformed into S. lividans, about 40-45% of the resulting colonies were able to produce clavulanic acid, as shown in FIG. 5.

EXAMPLES Example 1

[0064] Bacterial strains, vectors and growth conditions

[0065]Streptomyces clavuligerus NRRL 3585, Stretomyces jumonjinenisis NRRL 5741, Streptomyces lipmanii NRRL 3584, Streptomyces griseus NRRL 3851, Nocardia lactamdurans NRRL 3802 and Streptomyces cattleya NRRL 3841 were provided by the Northern Regional Research Laboratories, Peoria, Ill. Streptomyces antibioticus ATCC 8663 and Streptomyces fradiae ATCC 19609 were obtained from the American Type Culture Collection. Streptomyces lividans strains 1326 and TK24 were provided by D. A. Hopwood (John Innes Institute, Norwich, U.K.), Streptomyces venezuelae 13s and Streptomyces griseofuscus NRRL B-5429 were obtained from L. C. Vining (Department of Biology, Dalhousie University, Halifax, N.S.). Cultures were maintained on either MYM (Stuttard (1982) J. Gen. Microbiol., v. 128, pp. 115-121) or on a modified R5 medium (Hopwood et al. (1985) in “Genetic Manipulation of Streptomyces : a laboratory manual”, John Innes Foundation, U.K.) containing maltose instead of glucose and lacking sucrose (R5-S). Escherichia coli MV1193 (Zoller and Smith (1987) Methods in Enzymology, v. 154, pp. 329-349), used as recipient for all of the cloning and subcloning experiments, was grown in Luria Broth (LB; Sambrook et al. (1989) in “Molecular Cloning: a laboratory manual”, Cold Spring Harbour, N.Y.) or on LB agar (1.5%) plates containing ampicillin (50 μg/mL) or tetracycline (10 μg/mL). The cloning vectors pUC118 and pUC119 (Vieira and Messing (1987) Methods in Enzymology, v. 153, pp. 3-11) were provided by J. Vieira (Waksman Institute of Microbiology, Rutgers University, Piscataway, N.J.). The plasmid vector pJOE829 was generously provided by J. Altenbuchner (University of Stuttgart, Stuttgart, Germany). The plasmid pIJ702 was obtained from the American Type Culture Collection. Restriction enzymes were purchased from Boehringer Mannheim, and used according to the manufacturers' specifications.

[0066] Separation of CLA from ACVS

[0067] CLA was previously characterized as a 32,000 Da molecular weight protein present in preparations of highly purified ACVS (Jensen et al. (1990), supra). The small size of CLA suggested that its co-purification with ACVS resulted from a physical association between the two proteins.

[0068] ACVS and CLA were resolved by applying a 0.2 ml sample of purified ACVS containing CLA onto a Superose 6 HR 10/30 (Pharmacia), which was equilibrated and eluted in 0.1 M MOPS buffer, pH 7.5 containing 0.05 M KCl, 1 mM dithiothreitol, and 20% glycerol, at a flow rate of 0.25 ml/min.

[0069] Comparison of the CLA retention time with those of molecular weight standards indicated that the native molecular weight of CLA was in excess of 270 kDa. The difference in molecular weight between native and denatured forms of CLA suggests that the native protein exists as an oligomer of eight identical subunits.

[0070] Isolation of gene (cla) for CLA

[0071] N-terminal amino acid sequence information for CLA was obtained by electrophoretically transferring the protein from SDS polyacrylamide gels onto Immobilon membranes (Millipore Ltd., ) and submitting the material to the Protein Microsequencing Laboratory (University of Victoria,) for analysis. Information obtained for 25 amino acids at the N-terminus (amino acid residues 1-25 of SEQ ID NO:6) was used to prepare a 24 mer oligonucleotide probe (SEQ ID NO:25) with 8-fold degeneracy to the amino acid sequence underlined in FIG. 1. The amino acids in brackets indicate ambiguities in the N terminal sequence. The actual DNA sequence from the cloned fragment is indicated in SEQ ID No: 1.

[0072] The probe was designed as an 8-fold degenerate mixture of oligonucleotides to take into consideration the biased codon usage of streptomyces (Wright and Bibb (1992), Gene, v. 113, pp. 55-65).). End-labelled probe was then used to screen a cosmid library of S. clavuligerus genomic DNA fragments.

[0073] A library of S. clavuligerus genomic DNA fragments (15-22 kb size fractionated fragments) was constructed as previously described (Doran et al. (1990), J. Bacteriol, v. 172, pp. 4909-4918). Using the cosmid vector pLAFR3. A collection of 1084 isolated E. coli colonies contained recombinant cosmids was screened for the presence of cla using the 24-mer mixed oligonucleotide probe (FIG. 1) which had been end-labelled with [γ-³²P]dATP and polynucleotide kinase (Boehringer Mannhein). Colony hybridization and subsequent washing was performed as described by Sambrook et al., (1989), at 55° C. with a fmal wash in 0.2X SSC (IX SSC, 0.15M NaCl and 0.015M sodium citrate) and 0.1% SDS.

[0074] Five colonies which gave strong hybridization signals were isolated from the panel of 1084 clones, and restriction analysis showed that the positive clones contained overlapping fragments of DNA. Two clones, K6L2 and K8L2, with sequences that spanned about 40 kb of the S. clavuligerus genome, were chosen for further analysis. Clone K8L2 contained about 22 kb of S. clavuligerus genomic DNA and included a portion of cla and all of the pcbC gene which encodes IPNS in the penicillin/cephamycin biosynthetic pathway. A restriction map of K6L2 is shown in FIG. 7. Within the approximately 27 kb of DNA contained in K6L2, the oligonucleotide probe hybridized to a 2.0 kb NcoI fragment which was subsequently found to contain the entire cla gene. Hybridization studies, restriction mapping and DNA sequence analysis revealed that cla was situated 5.67 kb downstream of the pcbC gene of S. clavuligerus (FIG. 7).

[0075] DNA sequencing and analysis

[0076] Ordered sets of deletions were generated (Henikoff, 1984) extending across the cla region of the 2.0 kb NcoI fragment (FIG. 7(C)). The deletion generated fragments were sequenced in both orientations by the dideoxynucleotide chain termination method of (Sanger et al. (1977), P.N.A.S., v. 74, pp. 5463-5467) using Sequenase (version 2.0) DNA polymerase (United States Biochemical Corporation). Areas of compression in the sequence band pattern were relieved by carrying out reactions using 7-deaza-dGTP in place of dGTP. The nested deletion fragments resided either in pUC118 or pUC119, and were sequenced using the commercially available universal primers.

[0077] The nucleotide sequence data were analyzed for the presence of restriction sites, open reading frames (ORFs) and codon usage by the PC-Gene programme (Intelligenetics Corp.). Similarly searches were accomplished with the FASTA program searching the GenPept database (release number 71) available through GenBank (Pearson and Lipman (1988), P.N.A.S., v. 85, pp. 2444-2448).

[0078] An ORF at 939 bp with a potential ribosome site 9 bp from the GTG start codon was found which encoded a putative protein with a molecular weight of 33,368 Da. This value is in close agreement to the molecular weight estimated for CLA by SDS-PAGE (Jensen et al., 1990). The analysis of percent G+C as a function of codon position (FRAME analysis), using the algorithm of Bibb et al., (1984), indicated the presence of a typical streptomycete ORF (data not shown) with a G+C content of 70%. Computer aided data base searches for sequences similar to cla revealed a high degree of similarity to agmatine ureohydrolase (40.5% identity over 291 amino acids) and somewhat lower similarity to arginases (29.6% identity over 135 amino acids to arginases from yeast and rat). The S. clavuligerus CLA sequence was aligned with the E. coli AUH sequence by the FASTA program described above. The AUH sequence had previously been aligned with the three ARG sequences (Szumanski & Boyle (1990), J. Bacteriol., v. 172, pp. 538-547). Identical matches in two or more sequences are indicated with upper case letters.

Example 2

[0079] DNA hybridization

[0080] Genomic DNA preparations from various Streptomyces species were isolated as described by Hopwood et al. (1985). For interspecies DNA hybridization analysis, 2.0 μg amounts of genomic DNA preparations were digested with NcoI for 16 h, and electrophoresed in 1.0% agarose gels. The separated DNA fragments were then transferred onto nylon membranes (Hybond-N, Amersham) and hybridized with a cla specific probe prepared by labelling an internal 459 bp SalI fragment (FIG. 1) with [α³²P]DAPT by nick translation. Hybridization was done as described by Sambrook et al., (1989). Hybridization membranes were washed twice for 30 min in 2X SSC; 0.1% SDS and once for 30 min in 0.1X SSC; 0.1% SDS at 65° C.

[0081] Sequences homologous to cla in other Streptomycetes

[0082] Three of six producers of β-lactam antibiotics, S. clavuligerus, s. lipmanii and S. jumonjinesis showed positive hybridization signals whereas S. cattleya, S. griseus, and N. lactamdurans did not (data not shown). None of the nonproducing strains examined, S. venezuelae, S. lividans, S. fradiae, S. antibioticus and S. griseofuscus gave any signal. All of the streptomycetes that gave positive signals were producers of clam-type metabolites (Elson et al., 1987).

Example 3

[0083] Disruption of the genomic cla gene

[0084] A 2.0 kb NcoI fragment that contained the entire cla gene was digested at its unique KpnI site and the ends made blunt by treatment with the Klenow fragment of E. coli DNA polymerase I. A thiostrepton resistance gene (tsr), isolated as a 1085 bp BclI fragment from pIJ702 and cloned into the BamHI site of pUC118 was excised as a SmaI/XbaI fragment and the ends made blunt as above and ligated into the KpnI site of cla. The ligation mixture was introduced into E. coli MV1193 and the transformants screened for the presence of the tsr gene by colony hybridization (Sambrook et al., 1989).

[0085] Replacement of the chromosomal cla gene by a copy disrupted by the insertion of tsr, at an internal KpnI site, was achieved by double recombination. Successful gene replacement was apparent when the 2.0 kb NcoI fragment which carries cla in the wild type organism was replaced by a 3.0 kb NcoI fragment due to the insertion of the 1.0 kb tsr gene in the mutants. Four of the five mutants tested showed the expected increase in the size of the NcoI fragments, and the larger NcoI fragments also hybridized with a tsr specific probe. The fifth mutant was apparently a spontaneous theostrepton resistant mutant.

[0086] Antibiotic Assay

[0087] The agar diffusion assay was used for determining both penicillin/cephamycin and clavulanic acid production. S. clavuligerus strains to be assayed were grown in 10 ml. amounts of Trypticase Soy Broth (TSB; Baltimore Biological Laboratories) medium with 1.0% starch for 48 h. The cultures were washed twice with 10.3% sucrose and once with MM (Jensen et al. (1982), J. Antibiot., v. 35, pp. 483-490) and the mycelium resuspended in 10.0 mL of MM. Two milliliters of washed cell suspension was inoculated into 100 mL of MM and incubated at 28° C. for 48 h. The cultures were harvested by centrifugation, and the supernatants were assayed for both penicillin/cephamycin and clavulanic acid using bioassay procedures described previously (Jensen et al. (1982), supra).

[0088] All of the resulting colonies with disrupted cla genes grew equally well on minimal medium and complex media and produced as much penicillin and cephamycin as did the wild-type, but produced no clavulanic acid (data not shown). HPLC analysis of cell supernatants confirmed the inability of the disrupted cla mutants to synthesize any clavulanic acid (data not shown).

Example 4

[0089] Protoplast formation and transformation

[0090]E. coli competent cell preparation and transformation were as described by Sambrook et al., (1989). Protoplasts of S. clavuligerus were, prepared, transformed and regenerated as described by Bailey et al. (1984), Bio/Technology, v. 2, pp. 808-811, with the following modifications. Dextrin and arginine in the regeneration medium were replaced by starch and sodium glutamate respectively. Protoplasts were heat shocked at 43° C. for 5 min prior to the addition of DNA. Standard procedures were used for protoplasting and transformation of S. lividans (Hopwood et al. (1985)).

[0091] The 11.6 kb EcoRI fragment from K6L2 (FIG. 7) was cloned into the EcoRI site of pCAT-119. pCAT-119 is derivative of pUC119 which was prepared by insertionally inactivating the ampicillin resistance gene of pUC119 by the insertion of a chloramphenicol acetyltransferase gene (Jensen et al. (1989), Genetics & Molec. Biol. of Ind. Microorg., pp. 239-245 Ed. Hershberger, Amer. Soc. Microbiol). The PCAT-119 plasmid carrying the 11.6 kb fragment was then digested with PstI and ligated to the Streptomyces plasmid pIJ702, which had also been digested with PstI. The resulting bifunctional plasmid carrying the 11.6 kb insert was capable of replicating in either E. coli (with selection for chloramphenicol resistance) or in S. lividans (with selection for thiostrepton resistance). The ligation mixture was transformed to E. coli. Plasmid DNA was isolated from several of the chloramphenicol resistant transformants and analyzed by agarose gel electrophoresis to ensure that the proper plasmid construct was obtained. This isolated plasmid material from E. coli was then transformed into S. lividans as described by Hopwood and transformants were selected by plating onto R2YE medium containing thiostrepton at a concentration of 50 μg/ml.

[0092] Thiostrepton resistant S. lividans transformants carrying the bifunctional plasmid with the 11.6 kb insert were patched onto MYM agar plates and allowed to incubate for 48 h at 28° C. before they were overlayered with molten soft nutrient agar containing penicillin G at a concentration of 1 μg/ml and inoculated with Staphylococcus aureus N-2 as indicator organism (Jensen, 1982). (S. aureus N-2. was obtained form the Department of Microbiology Culture Collection, University of Alberta. Any organism which produces a β-lactamase sensitive to clavulanic acid may be used as indicator organism.) Zones of inhibition which appeared around the S. lividans colonies upon incubation overnight at 30° C. were evidence of clavulanic acid production. Clavulanic acid-producing colonies were found amongst these initial S. lividans transformants at a frequency of about 12%. When plasmid DNA was isolated from one of these clavulanic acid-producing transformants and re-introduced into S. lividans, the frequency of clavulanic acid production in these 2nd round transformants was about 40-45%. FIG. 5 shows a photograph of an agar plate bearing 2nd. round transformants. Zones of inhibition are seen as clear areas in the agar; these appear on the photograph as dark circular areas.

Example 5

[0093] Sequencing of 15 kb DNA fragment

[0094] Ordered sets of deletions were generated as described in Example 1 using fragments of the DNA insert from the cosmid clone K6L2 (FIG. 7) and subcloned into the E. coli plasmids pUC118 and pUC119. Overlapping fragments were chosen which extended from the end of the pcbC gene downstream for a distance of about 15 kb ending at the BglII site. The deletion generated fragments were sequenced in both orientations as described in Example 1. The sequence is shown in SEQ ID NO:1.

Example 6

[0095] Gene Disruption of the ORFs Contained within the 12 kb Fragment

[0096] Gene disruption experiments were conducted on the various open reading frames (ORF) to determine if a particular ORF was involved in clavulanic acid production or not. In order to determine the role of the various ORFs in clavulanic acid biosynthesis, mutants disrupted in one of the ORFs were constructed by a gene replacement procedure based on that described in Paradkar and Jensen (Journal of Bacteriology, March 1995, Vol. 177, no. 5, pages 1307 to 1314).

[0097] In all cases, either an apramycin-resistance gene cassette (apr), or a neomycin resistance gene cassette (neo) was used to disrupt the genes encoded within the 12-kb DNA fragment. Before use, unless otherwise indicated, the apr-cassette was modified by adding NcoI restriction sites to both ends. This modification of the apr-cassette has been described in Paradkar and Jensen (Journal of Bacteriology, March 1995, Vol. 177, no. 5, pages 1307 to 1314). Since four NcoI sites are present within the 12 kb fragment and present within ORFs 3, 5, 6, and 8, respectively, insertion of the apr-cassette within the NcoI sites created a series of plasmids (called pCATL2) with disruptions in ORFs 3, 5, 6, or 8. The plasmids containing these fragments are referred to as pCATL2orf3, pCATL2orf5, pCATL2orf6, and pCATL2orf8, respectively. The construction of pCATL2orf5 has been previously disclosed (Paradkar and Jensen, Journal of Bacteriology, March 1995, Vol. 177, no. 5, pages 1307 to 1314). From each of these plasmids, a smaller fragment carrying only the disrupted gene and some flanking sequence (see details below) was subcloned and finally inserted into the Streptomyces vector pIJ486 (obtained from David A Hopwood, John Innes Institute) for transformation into S. clavuligerus. All routine manipulations involved in subcloning were done in Escherichia coli using standard cloning vectors, such as pUC119, pBluescript SK + etc. Since ORF2, ORF7 and ORF9 do not contain NcoI sites, the disruptions of these ORFs were done in a slightly different manner. The preparation of ORF2, ORF3, ORF6, ORF7, ORF8 and ORF9 disruption mutants are described in more detail below.

[0098] Construction of the Insertional Mutants

[0099] 1. ORF2 Mutant

[0100] NotI-NcoI linker oligonucleotides were added to both ends of the apr gene cassette, and then this modified apr gene fragment was inserted into the NotI site within ORF2 carried on a 2.1-kb EcoRI-BglII fragment. Subsequently, the EcoRI-BglII fragment carrying the disrupted ORF2 was inserted into pIJ486.

[0101] Genomic DNA isolated from the wild type (wt) and from an ORF2 mutant was digested with EcoRI and NcoI, and then probed with a 2.1-kb EcoRI-BglII fragment (FIG. 18). The 2.7 kb hybridizing fragment present in the wild type has been replaced with a 1.8 kb fragment in the mutant. A 0.85 kb fragment is also expected to hybridize to the probe in the mutant, but owing to the conditions of electrophoresis under which the gel was that smaller fragment was not retained on the gel. The sizes of the remaining hybridizing fragments are consistent with the replacement of the wild type ORF2 with the apr-disrupted ORF2.

[0102] 2. ORF3 Mutant

[0103] The apr cassette (with NcoI sites on each end) was inserted into the NcoI site within ORF3 carried on a 4 kb EcoRI-KpnI fragment. The fragment carrying the disrupted ORF3 was then subcloned into pIJ486.

[0104] Genomic DNA from the wild type (wt) and from an ORF3 mutant was digested separately with BglII, and with KpnI, and probed with a 0.6-kb BglII-NcoI fragment (FIG. 19). In the BglII digests, the 6.5 kb hybridizing fragment present in the wild type has been replaced with 8 kb fragment in the mutant, while in the KpnI digests, the 5.8 kb-fragment has been replaced with a 4.5 kb fragment. The sizes of these hybridizing fragments are consistent with the replacement of the wild type ORF3 with the apr-disrupted ORF3.

[0105] 3. ORF6 Mutant

[0106] An 8-kb BglII fragment carrying the disrupted ORF6 was subcloned from pCATL2orf6 into pIJ486.

[0107] Genomic DNA from the wild type (wt) and from an ORF6 mutant was digested with BglII, and probed with a 1.6-kb NruI fraginent (FIG. 20). The 6.5 kb hybridizing fragment present in the wild type has been replaced with an 8 kb fragment in the mutant. The sizes of these hybridizing fragments are consistent with the replacement of the wild type ORF6 with the apr-disrupted ORF6.

[0108] 4. ORF7 Mutant

[0109] The NruI site from the polylinker of the plasmid vector pSL1180 (Pharmacia) was eliminated by digesting with enzymes flanking the NruI site and religating the resulting plasmid. A 1.9 kb BglII-NcoI fragment of S. clavuligerus DNA encompassing the ORF7 gene was then cloned into the modified pSL1180 plasmid vector. The neomycin resistance cassette was removed from pFDNEO-S (Denis, F., and R. Brzezinski. 1991. An improved aminoglycoside resistance gene cassette for use in Gram-negative bacteria and Streptomyces. FEMS Microbiol. Lett. 81:261-264), made blunt-ended by treatment with the Klenow fragment of DNA polymerase, and then introduced into the NruI site of the cloned ORF7 gene. The Streptomyces plasmid pJOE829 (Aidoo et al., 1994, Gene 147: 1-46.) was ligated to the vector carrying the disrupted ORF7 gene to allow the creation of a shuttle vector. This gene disruption construct was then used to generate an ORF7 mutant using a gene replacement procedure based on that described in Paradkar and Jensen (Journal of Bacteriology, 1995, 177:1307-1314.)

[0110] Putative ORF7 disruptant mutants were selected on the basis of their neomycin resistant, hygromycin sensitive phenotype. The identity of the mutants was confirmed by Southern analysis. Genomic DNA from the wild type (wt) and the orf7 mutant was digested with ApaI, and probed with a 1.9 kb NcoI+BglII fragment (FIG. 21). Both the wt and the orf7 mutant gave two hybridizing ApaI bands. The mutant (FIG. 21 lane 3) gave a 1 kb and a 2.8 kb band whereas the wt (FIG. 21 lane 2) gave a 1 kb and a 1.8 kb band. The sizes of the hybridizing fragments are consistent with the replacement of the wild type orf7 with the neo-disrupted orf7.

[0111] 5. ORF8 Mutant

[0112] A 2.9 kb BglII-EcoRI fragment carrying the disrupted ORF8 was subcloned from pCATL2orf8 into pIJ486.

[0113] Genomic DNA from the wild type (wt) and from an ORF8 mutant was digested with BglII, and also with both BglII and KpnI, and probed with a 1.3 kb BglII-NcoI fragment (FIG. 22). In the BglII digests, the 1.9 kb hybridizing fragment present in the wild type has been replaced with a 3.4 kb fragment in the mutant, while in the BglII/KpnI digests, the 1.9-kb fragment has been replaced with a 1.3 kb fragment. The sizes of these hybridizing fragments are consistent with the replacement of the wild type ORF8 with the apr-disrupted ORF8.

[0114] 6. ORF9 Mutant

[0115] The apr gene cassette was first cloned as an EcoRI-PstI fragment into the E. coli vector Pbluescript, and re-isolated as an EcoRV-SmaI fragment. This fragment was then inserted into the MscI site present in ORF9 contained within a 2.4 kb BglII fragment. Subsequently, the 4 kb fragment carrying the disrupted ORF9 was inserted into pIJ486.

[0116] Genomic DNA from the wild type (wt) and an ORF9 mutant was digested with BglII, and with both BglII and EcoRI, and probed with a 0.6-kb BglII-SstI fragment (FIG. 23). In the BglII digests, the 2.4 kb hybridizing fragment present in the wild type has been replaced with a 3.9 kb fragment in the mutant, while in the BglII-EcoRI digests, the 1-kb fragment has been replaced with a 0.6 kb fragment. In BglII-EcoRI digests of the ORF9 mutant genomic DNA another 3.3-kb EcoRI-BglII fragment might have been expected to hybridize to the probe, but failure to see this fragment can be attributed to the fact that the probe has only approximately 50 bp of sequence homologous to the expected fragment. Since the post-hybridization washes were carried out under stringent conditions this small amount of homology might not be sufficient to give a hybridizing band. Nevertheless, the sizes of other hybridizing fragments are consistent with the replacement of the wild type ORF9 with the apr disrupted ORF9.

[0117] The Effect of Mutations Within the ORF's of the 12 kb Fragment on Clavulanic Acid Synthesis

[0118] 1. Bioassay

[0119] The bioassay used to monitor clavulanic acid production in the gene disruption mutants has already been described above in Example 3 under the heading Antibiotic assay. The results are summarized in Table II below: TABLE II Strain No Penicillin Penicillin G (1μ/ml) wild type 0 mm 28 mm  ORF2 0 mm 0 mm ORF3 0 mm 0 mm ORF6 0 mm 0 mm ORF7 0 mm 0 mm ORF8 0 mm 0 mm ORF9 0 mm 0 mm

[0120] If a zone of inhibition is obtained in the presence of penicillin but not in the absence thereof or if the size of the zones in the presence of penicillin is greater than the zones without penicillin, it is indicative of a β-lactamase inhibitory activity of clavulanic acid. The bioassay thus showed that transformants containing mutants with insertion in any one of the ORF2, ORF3, ORF6, ORF7, ORF8 or ORF9 failed to produce β-lactamase inhibitory activity of clavulanic acid, thus indicating that each of these ORFs are involved in the production of clavulanic acid.

[0121] 2. HPLC Analysis

[0122] The amount of clavulanic acid produced by each transformant was also quantitated in each sample by HPLC. This assay was done to confirm the presence or absence of clavulanic acid production, within the cultured supernatants produced from each gene disruption mutant.

[0123] Culture Conditions for the Growth of Wild Type and the Mutants for Clavulanic Acid Production

[0124] Spores of transformants containing insertional mutations within either ORF2, ORF3, ORF6, ORF8 or ORF9 were first inoculated into 20 mL Trypticase Soy Broth containing 1% starch, and the culture was grown shaking for 48 hours at 28° C. which served as a seed culture. Seed cultures were then used at 1% inoculum to inoculate 20 ml Starch Asparagine medium the composition of which has been described earlier (Paradkar and Jensen (1995), J Bacteriol Vol. 177, pp. 1307-1314), and the culture was grown in the same conditions as the seed culture. For ORF7 cultures, spores were inoculated into 25 ml Trypticase Soy broth containing 1% maltose to produce the seed culture, which was used at 2% inoculum to inoculate 25 ml Starch Asparagine medium as outlined above. Supernatants (0.1 mL) were obtained from 96 hour cultures, and derivatized with 25 μL of imidazole reagent. Subsequently, a 50 μL aliquot of the derivatized sample was analyzed by HPLC.

[0125] The high pressure liquid chromatography assay was conducted as described in Foulstone and Reading (1982, Antimicrob. Agents Chemother. 22:753-762).

[0126] Analysis was performed with a model M-45 pump, model 712 WISP automated sample injector, and model 480 variable-wavelength UV detector, all from Millipore Waters (Mississauga, Ontario, Canada). Samples (100 μl) were mixed with imidazole reagent (25 μl) and incubated at room temperature for 15 min to form imidazole-derivatized clavulanic acid. Derivatized samples (50 μl) were analyzed on a reverse-phase column (μBondapak-C18) with an isoctatic buffer system consisting of 0.1 M KH₂PO₄-6% methanol, PH 3.2 (adjusted with H₃PO₄).

[0127] The HPLC chromatograms of culture supernatants from transformants containing an insertional mutation within ORF2, ORF3, ORF6, ORF7, ORF8 or ORF9 and of the wild type were obtained. An example of these results is illustrated in FIG. 24, which represents HPLC profiles of wild type (FIG. 24 (A) wild type control for ORF7, or FIG. 24 (D), wild type control for ORF8) and ORF7 (FIG. 24 (B), or the ORF8 (FIG. 24 (E)) insertion mutants. As can be seen from FIG. 24, clavulanic acid was detected in the supernatant of the wild type as a peak, at a retention time of approximatelyl 10.13 minutes (FIG. 24 (B)) or 9.1 minutes (FIG. 24 (C)). An authentic sample of clavulanic acid, obtained just prior to the analysis of the ORF8 data also gave a retention time of approximately 9.1 minutes (FIG. 24 (C)). The amount of clavulanic acid produced by the wild type was determined by integrating the clavulanic acid peak in the wild type supernatant. No clavulanic acid was detected in the supernatants of any of the insertion mutants.

[0128] All citations are incorporated herein by reference. The present invention is not limited to the features of the embodiments described herein, but includes all variations and modifications within the scope of the claims.

1 25 1 15120 DNA Streptomyces clavuligerus 1 gcggaaccgg ccgcccctga gcggggcggc cgggaaggaa acgggccggt cgtcccctcg 60 ggagggggcg gccggcccgt ccggtgcgcg cggtgggtgc ggcgcgggtc agccggccgc 120 gaggttgctg aggaacttcg cggcgacggg gcccgcgtcg gcgccgcccg acccgccgtc 180 ctccagcagg accgaccagg cgatgttccg gtcgccctgg tagccgatca tccaggcgtg 240 cgtcttcggc ggcttctcgg tgccgaactc ggcggtaccg gtcttggcgt gcggctgtcc 300 gccgaggccc cgcagggcgt cgccggcgcc gtcggtgacg gtcgaacgca tcatggaacg 360 cagcgagtcg acgatgcccg gggccatccg gggggcctgg tgcggcttct tgaccgcgtc 420 gggcaccagc acgggctgct tgaactcgcc ctgcttgacg gtggcggcga tggaggccat 480 caccaggggc gacgcctcga ccctggcctg tccgatggtg gacgcggcct tgtcgttctc 540 gctgttggag acggggacgc tgccgtcgaa ggtggaggcg ccgacgtccc aggtgccgcc 600 gatgccgaag gcttcggcgg cctgcttcag gctggactcg gagagcttgc tgcgggagtt 660 gacgaagaac gtgttgcagg agtgggcgaa gctgtcccgg aaggtcgagc ccgcgggcag 720 cgtgaactgg tcctggttct cgaagctctg gccgttgaca tgggcgaact tcgggcagtc 780 ggcccgctcc tccgggttca tcccctgctg gagcagggcc gcggtggtga ccaccttgaa 840 ggtggagccg ggcgggtagc ggccctccag cgcgcggttc atgccggagg gcacgttcgc 900 ggcggccagg atgttgccgg tggcggggtc gacggcgacg atcgccgcgt tcttcttcga 960 gccctccagg gccgccgcgg cggcggactg gacccgcggg tcgatggtgg tcttcaccgg 1020 cttgccctcg gtgtccttga ggccggtgag cttcttgacc acctggccgg actcacggtc 1080 caggatcacg accgagcgcg ccgcgccgga gccgccggtg agctgcttgt cgtagcggga 1140 ctggaggccc gccgagccct tgccggtcct ggggtcgacc gcgccgatga tggaggcggc 1200 ctggaggaca ttgccgttgg cgtcgaggat gtccgcgcgc tcccgcgact tgagggcgag 1260 ggtctgcccc ggaaccatct gcggatggat catctcggtg ttgaacgcga ccttccactc 1320 cttgccgccg ccgacgacct tcgcggtgga gtcccaggcg tactccccgg ccccggggag 1380 ggtcattctg acggtgaacg gtatctccac ctcgccctcg gggttcttct ccccggtctt 1440 ggcggtgatc tccgtcttcg tcggcttgag gttggtcatg acggatttga tcagcgactc 1500 ggcgttgtcc ggggtgtccg tcagcccggc ggccgtcggg gcgtcgccct tctcccaggc 1560 gccgaggaag gtgtcgaact gtccggccgc cgcctccacc tcggggtcgc ccgaatcctt 1620 ctcgtcggca accaggctgg tgtaacccca atagccgagc cccaccgtca cggccagccc 1680 ggcgaccacc gcggtggccg cccggccacg ggagcggcgc ctgccctgcg gcgggtcatc 1740 gccatagttg tcggaatgcg tcatggggcc aggctatgcg ggcgccctct ttccctcctc 1800 cccggatacc gcgtttcagg acagtcaagg ggccgaacgg agggctggac cagccgctca 1860 gcggcccgtt cccacccctt ggggggaagc ggcacccgga aggtgaccga ggcaacatcc 1920 atggaaaggg gagcgaatcg gtcgccgagt tcaccgcgat tggagtagac ctctgaaagc 1980 gtgacagcgg ggagtagcga caaaacggtc agacccctga agggaattga ctgaattcca 2040 tcgattgatt gtgcgaatcc agggaagccg agcgaattcg agtcatcggg ttcggcgacg 2100 gatgggcggt tcggccacgc accgtcactc ttcgtcccct cttcacaaga actcccgata 2160 cgtggagaag agagcgtgaa gagcgcgtcc ggtcagggtt gccgagaacc gtccaccatg 2220 acggagcctg gtactgacgg agtctggaga ccgctcatgt cccgtgtatc gaccgccccc 2280 agcggcaagc ctaccgccgc tcacgccctc ctgtcacggt tgcgtgatca cggtgtgggg 2340 aaggtgtttg gggttgtcgg ccgagaggcc gcgtcgattc tcttcgacga ggtcgagggg 2400 atcgacttcg ttctgacccg ccacgagttc accgcgggtg tcgccgctga tgtcctcgcg 2460 cggatcaccg gtcgccccca ggcgtgctgg gccaccctgg gccccggtat gaccaacctc 2520 tccaccggta tcgccacgtc cgtcctggac cgctcgccgg tcatcgcgct cgccgcgcag 2580 tcggagtcgc acgacatctt cccgaacgac acccaccagt gcctggactc ggtggcgatc 2640 gtcgccccga tgtccaagta cgccgtggag ctccagcggc cccacgagat caccgacctc 2700 gtcgactccg ccgtgaacgc ggccatgacc gagccggtcg ggccctcctt catctccctc 2760 ccggtggacc tgctcggctc ctccgagggc atcgacacca ccgtccccaa cccgccggcg 2820 aacaccccgg cgaaaccggt cggcgtcgtc gccgacggct ggcagaaggc cgccgaccag 2880 gccgccgccc tgctcgccga ggccaagcac ccggtgctcg tcgtcggagc ggccgcgatc 2940 cgctcgggcg ccgtcccggc gatccgcgcc ctggccgagc gcctgaacat cccggtcatc 3000 acgacctaca tcgccaaggg tgtcctgccg gtcggccacg agctgaacta cggcgccgtc 3060 accggctaca tggacggcat cctcaacttc ccggcgctcc agaccatgtt cgccccggtg 3120 gacctcgtcc tcaccgtcgg ctacgactac gccgaggacc tgcgcccgtc catgtggcag 3180 aagggcatcg agaagaagac cgtccgtatc tccccgacgg tcaacccgat cccccgggtc 3240 taccggcccg acgtcgacgt cgtcaccgac gtcctcgcct tcgtggagca cttcgagacc 3300 gcgaccgcct ccttcggggc caagcagcgc cacgacatcg agccgctgcg cgcccggatc 3360 gcggagttcc tggccgaccc ggagacctac gaggacggca tgcgcgtcca ccaggtcatc 3420 gactccatga acaccgtcat ggaggaggcc gccgagcccg gcgagggcac gatcgtctcc 3480 gacatcggct tcttccgtca ctacggtgtg ctcttcgccc gcgccgacca gcccttcggc 3540 ttcctcacct cggcgggctg ctccagcttc ggctacggca tccccgccgc catcggcgcc 3600 cagatggccc gcccggacca gccgaccttc ctcatcgcgg gtgacggcgg cttccactcc 3660 aacagctccg acctggagac catcgcccgg ctcaacctgc cgatcgtgac cgtcgtcgtc 3720 aacaacgaca ccaacggcct gatcgagctg taccagaaca tcggtcacca ccgcagccac 3780 gacccggcgg tcaagttcgg cggcgtcgac ttcgtcgcgc tcgccgaggc caacggtgtc 3840 gacgccaccc gcgccaccaa ccgcgaggag ctgctcgcgg ccctgcgcaa gggtgccgag 3900 ctgggtcgtc cgttcctcat cgaggtcccg gtcaactacg acttccagcc gggcggcttc 3960 ggcgccctga gcatctgatc atgggggcac cggttcttcc ggctgccttc gggttcctgg 4020 cctccgcccg aacgggcggg ggccgggccc ccggcccggt cttcgcgacc cggggcagcc 4080 acaccgacat cgacacgccc cagggggagc gctcgctcgc ggcgaccctg gtgcacgccc 4140 cctcggtcgc gcccgaccgc gcggtggcgc gctccctcac cggcgcgccc accaccgcgg 4200 tgctcgccgg tgagatctac aaccgggacg aactcctctc cgtgctgccc gccggacccg 4260 cgccggaggg ggacgcggag ctggtcctgc ggctgctgga acgctatgac ctgcatgcct 4320 tccggctggt gaacgggcgc ttcgcgaccg tggtgcggac cggggaccgg gtcctgctcg 4380 ccaccgacca cgccggttcg gtgccgctgt acacctgtgt ggcgccgggc gaggtccggg 4440 cgtccaccga ggccaaggcg ctcgccgcgc accgcgaccc gaagggcttc ccgctcgcgg 4500 acgcccgccg ggtcgccggt ctgaccggtg tctaccaggt gcccgcgggc gccgtgatgg 4560 acatcgacct cggctcgggc accgccgtca cccaccgcac ctggaccccg ggcctctccc 4620 gccgcatcct gccggagggc gaggccgtcg cggccgtgcg ggccgcgctg gagaaggccg 4680 tcgcccagcg ggtcaccccc ggcgacaccc cgttggtggt gctctccggc ggaatcgact 4740 cctccggggt cgcggcctgt gcgcaccggg cggccgggga actggacacg gtgtccatgg 4800 gcaccgacac gtccaacgag ttccgcgagg cccgggcggt cgtcgaccat ctgcgcaccc 4860 ggcaccggga gatcaccatc ccgaccaccg agctgctggc gcagctcccg tacgcggtgt 4920 gggcctccga gtcggtggac ccggacatca tcgagtacct gctccccctg acagcgctct 4980 accgggcgct cgacgggccg gagcgccgca tcctcaccgg gtacggcgcg gacatccccc 5040 tcgggggcat gcaccgcgag gaccggctgc ccgcgctgga caccgttctc gcgcacgaca 5100 tggccacctt cgacgggctg aacgagatgt ccccggtgct gtccacgctg gcggggcact 5160 ggaccaccca cccgtactgg gaccgggagg tcctcgatct gctggtctcg ctggaggccg 5220 ggctcaagcg gcggcacggc cgggacaagt gggtgctgcg cgccgcgatg gccgacgccc 5280 tcccggcgga gaccgtcaac cggcccaagc tgggcgtcca cgagggctcg ggcaccacgt 5340 cctcgttctc ccggctgctg ctggaccacg gtgtcgccga ggaccgcgtc cacgaggcga 5400 agcggcaggt ggtgcgcgag ctgttcgatc tcacggtcgg gggcggacgg cacccctccg 5460 aggtggacac cgacgatgtg gtgcgctccg tggccgaccg gaccgcgcgg ggggcggcct 5520 agtcccgcca cggggagccc gccggacgcc ggacccgcgc gggacccgta cccggggccg 5580 cccgcggact ccggcgcacc ggcacccctg tcccccaccc gttgacgacc gtcggccctc 5640 ggccctcgcg gcccctgacg accgtcgccc gattcccagg agggagctga aagcgtggag 5700 cgcatcgact cgcacgtttc accccgctac gcacagatcc ccaccttcat gcgcctgccg 5760 cacgatcccc agccccgcgg ctatgacgtg gtggtcatcg gagcccccta cgacgggggc 5820 accagctacc gtcccggcgc ccggttcggc ccccaggcca tccgcagtga gtcgggcctc 5880 atccacggtg tcggcatcga ccggggcccc ggcacgttcg acctgatcaa ctgtgtcgac 5940 gccggggaca tcaatctgac gccgttcgac atgaacatcg cgatcgacac ggcgcagagc 6000 catctgtcgg gcctgctgaa ggccaacgcc gcctttctga tgatcggcgg cgaccactcg 6060 ctgacggtgg ccgccctgcg cgcggtcgcg gagcagcacg gcccgctcgc cgtggtgcac 6120 ctggacgcgc actccgacac caacccggcc ttctacgggg gccggtacca ccacggcacc 6180 cccttccggc acgggatcga cgagaagctg atcgacccgg cggcgatggt ccagatcggc 6240 atccggggcc acaacccgaa gccggactcg ctcgactacg cccggggcca cggcgtccgg 6300 gtggtcacgg cggacgagtt cggcgagctg ggggtgggcg ggaccgccga cctcatccgc 6360 gagaaggtcg gccagcggcc cgtgtacgtc tcggtcgaca tcgacgtggt cgaccccgcc 6420 ttcgcccccg gtacgggcac gcccgcgccg ggcgggctcc tctcgcgcga ggtgctggcg 6480 ctgctgcgct gcgtgggtga cctgaagccg gtcggcttcg acgtgatgga ggtgtcaccc 6540 ctctacgacc acggcgggat cacttcgatc ctggccacgg agatcggtgc ggaactgctc 6600 taccagtacg cccgagccca cagaacccag ttgtgaagga gacatcgtgt catggcctct 6660 ccgatagttg actgcacccc gtaccgcgac gagctgctcg cgctcgcctc cgagcttccc 6720 gaggtgccgc gcgcggacct ccatggcttc ctcgacgagg cgaagacgct ggccgcccgt 6780 ctcccggagg ggctggccgc cgctctcgac accttcaacg ccgtgggcag cgaggacggt 6840 tatctgctgc tgcgcgggct gcccgtcgac gacagcgagc tgcccgagac gccgacctcc 6900 accccggccc cgctggaccg caagcggctg gtgatggagg ccatgctcgc gctggccggc 6960 cgccggctcg gtctgcacac ggggtaccag gagctgcgct cgggcacggt ctaccacgac 7020 gtgtacccgt cgcccggcgc gcactacctg tcctcggaga cctccgagac gctgctggag 7080 ttccacacgg agatggcgta ccacatcctc cagccgaact acgtcatgct ggcctgctcc 7140 cgcgcggacc acgagaaccg ggcggagacg ctggtcggct cggtccgcaa ggcgctgccc 7200 ctgctggacg agaagacccg ggcccgtctc ttcgaccgca aggtgccctg ctgcgtggac 7260 gtggccttcc gcggcggggt cgacgacccg ggcgcgatcg ccaacgtcaa gccgctctac 7320 ggggacgcga acgacccgtt cctcgggtac gaccgcgagc tgctggcgcc ggaggacccc 7380 gcggacaagg aggccgtcgc ccatctgtcc caggcgctcg acgatgtgac cgtcggggtg 7440 aagctcgtcc ccggtgacgt cctcatcatc gacaacttcc gcaccacgca cgcgcggacg 7500 ccgttctcgc cccgctggga cgggaaggac cgctggctgc accgcgtcta catccgcacc 7560 gaccgcaatg gacagctctc cggcggcgag cgcgcgggcg acaccatctc gttctcgccg 7620 cgccgctgag cccggctccc cgaggccctg ggccccggcg ccggaaccgg ctcccggtcc 7680 tgccccctca cccgccgcgc gggtgagggg gcaggcccct ttgtgccggg tgccgtgcgt 7740 cctgcgaggg tgccggggcg ggggggacgg cggaggtgcc cggcggccgg gtgccgtgcg 7800 ccgcccgtgg gtgctgtaca gcactccgtg tgccgtgcgc caccccgtgc ataaatttgc 7860 cactctatgg gaaataatgc agagtgcgac gggtgaggcc gtcgccgtgc cctttccgtg 7920 acaggagacg ctgacatgtc cgacagcaca ccgaagacgc cccggggatt cgtggtgcac 7980 acggcgccgg tgggcctggc cgacgacggc cgcgacgact tcaccgtcct cgcctccacc 8040 gccccggcca ccgtgagcgc cgtcttcacc cgctcccgct tcgccgggcc gagcgtcgtg 8100 ctgtgccggg aggcggtggc cgacgggcag gcgcgcggtg tggtggtgct ggcccgcaac 8160 gcgaatgtcg cgaccggcct ggagggcgag gagaacgcgc gcgaggtgcg cgaggccgtc 8220 gcccgggccc tcgggctgcc ggagggcgag atgctgatcg cctccaccgg ggtgatcggc 8280 cggcagtacc cgatggagag catccgggag cacctcaaga cgctggagtg gcccgccggg 8340 gagggcggct tcgaccgcgc ggcccgcgcc atcatgacga ccgacacccg gcccaaggag 8400 gtccgggtca gcgtcggcgg ggcgaccctc gtgggcatcg ccaagggcgt cggcatgctg 8460 gagcccgaca tggcgacgct gctgaccttc ttcgccacgg acgcccggct ggacccggcc 8520 gagcaggacc gcctcttccg ccgggtcatg gaccgcacct tcaacgcggt cagcatcgac 8580 accgacacct ccaccagcga cacggcggtg ctgttcgcca acggcctggc gggcgaggtc 8640 gacgccgggg agttcgagga ggcgctgcac acggcggcgc tggccctggt caaggacatc 8700 gcgagcgacg gcgagggcgc ggccaagctg atcgaggtcc aggtcaccgg cgcccgcgac 8760 gacgcccagg ccaagcgggt cggcaagacc gtcgtcaact ccccgttggt gaagaccgcc 8820 gtgcacggct gcgaccccaa ctggggccgg gtcgccatgg cgatcggcaa gtgctcggac 8880 gacaccgaca tcgaccagga gcgggtgacg atccgcttcg gcgaggtcga ggtctatccg 8940 ccgaaggccc ggggcgacca ggccgacgac gcgctgcggg ccgccgtcgc ggagcatctg 9000 cggggcgacg aggtggtcat cgggatcgac ctcgccatcg cggacggggc cttcaccgtc 9060 tacggctgcg acctcaccga gggctatgtc cggctgaact cggagtacac cacctgatcc 9120 ccggacaggg aacgggccgc cgccccgttc cctgtccgct cccgtcccgt gtggttatac 9180 cgaccgttcc ccggctatgc gcacgggacg gagcggcccc cgccgggccc cgcccggccg 9240 cacgatgagg ggcgatgcaa ggtgacgagg gcaggaggga catggagacc actcggtcga 9300 cgaccgcgga cgagggcttc gacgccgggg tacggggagt ggtcgcgccg accgacgccc 9360 cgggcgggac gctgcggctg gtccgcacgg acgacttcga ctcgctcgac cccggcaaca 9420 cgtactacgc ctacacctgg aacttcctcc ggctcatcgg ccggacgctg gtcaccttcg 9480 acaccgcgcc gggcaaggcg ggccagcggc tcgtgcccga cctcgccgag tcgctgggcg 9540 agtcctccga ggacggccgg gtctggacct accggctgcg cgagggcctg cgctacgagg 9600 acggcacgcc ggtcgtctcg gccgacatca agcacgccat cgcccgcagc aactacggca 9660 ccgatgtcct gggcgccggt ccgacctact tccgccacct cctgggcacc gagtacggcg 9720 gcccctggcg ggagccggac gccgacggac cggtgacgct ggagaccccg gacgagcgga 9780 cgctggtctt ccggctgcgg gagccgttcg cggggatgga tctgctggcg accatgccgt 9840 ccaccacccc cgtgccgcgc gaccgggaca ccggcgccga gtaccggctg cggcccgtgg 9900 cgaccggccc gtaccggatc gtctcgtaca cccggggcga gctggccgtc ctggagccca 9960 atccgcactg ggaccccgag accgacccgg tgcgcgtcca gcgcgcctcc cggatcgagg 10020 tgcacctcgg caaggacccg cacgaggtgg accgcatgct gctggcgggc gaggcccatg 10080 tggacctcgc gggcttcggt gtgcagcccg cggcccagga gcgcatcctc gccgagccgg 10140 agctgcgcgc gcacgcggac aacccgctga ccggcttcac ctggatctac tgcctgtcga 10200 gccggatcgc cccgttcgac aatgtgcact gccggcgggc cgtgcagttc gccaccgaca 10260 aagcggccat gcaggaggcg tacggcggcg cggtgggcgg cgacatcgcg accaccctgc 10320 tgcccccgac cctcgacggc tacaagcact tcgaccgcta cccggtcggc cccgagggca 10380 ccggcgacct ggaggccgcc cgcgccgagc tgaagctggc cgggatgccc gacggcttcc 10440 gcaccaggat cgccgcccgc aaggaccggc tcaaggagta ccgggccgcc gaggcgctgg 10500 ccgccgggct cgcccgggtc ggcatcgagg cggaggtgct ggacttcccg tcgggcgact 10560 acttcgaccg ctacggcggc tgcccggagt atctgcgcga gcacgggatc gggatcatca 10620 tgttcggctg gggcgccgac ttccccgacg gatacggctt cctccagcag atcaccgacg 10680 ggcgcgcgat caaggagcgc ggcaaccaga acatgggcga gctggacgac ccggagatca 10740 acgcgctgct ggacgagggg gcgcagtgcg ccgacccggc gcggcgcgcg gagatctggc 10800 accgcatcga ccagctcacg atggaccacg cggtcatcgt tccgtatctg tacccgcggt 10860 ccctgctcta ccggcacccg gacacccgca acgccttcgt caccggctcc ttcgggatgt 10920 acgactacgt ggcgctcggc gcgaagtgag cacggggtcc ggccccggga ccgtatgtcc 10980 cggggccgga ccccgcccgt tccccgcccg gtccggtccg gacccggtcg cggcccgctc 11040 agccggacat ccgggccccg gccgcgaccc cgcgccggat cggccagtgg ccctgcgcca 11100 ggggccgttc cacgctgcgg caggcgagag cggcctcgcg gaactccgcc tcgtacagcg 11160 cgagctggcg caggaactgc cgggtcgggc cggtcaggct ggtcccccgc gggctgcgca 11220 gcagcagccg ggcgccgagg gactgctcca gccggtgaat ccggcgggtg agcgccgact 11280 ggctgatcga cagcaccgcc gcggcccggt tgatgctgcc gtgccgggcc acggcctgga 11340 gcagatggag atcgtccaca tccagtttgc ggccctcggc ctggccgggc acggagccct 11400 ggtcgggtcc cgccccgaag cggcgggcgt ccgcgccggt gcgctccgcg taccactgcg 11460 cccaccaggg ctcgtccagc aggtcgcggt ggtgttcggc gaagcgccgg agctggacct 11520 cggcgatcag cgcggccagc cgtcccgcca gcgcccgggg cacgatggtg gggtcgacga 11580 gcagactcgt ggtgcggcgc gggcgctccg ccagggagcg gcgcaccagc gaggggtcct 11640 gcaccgccgg gtgggtgggc gagccgagac ctatcgcgtc cccgcggcgc aggatgcccc 11700 gggcaaccga tgcccccgtg atgtggagcc gggtgggcgc ggtgagcccg gccagctgga 11760 agacacgtgt caccaggatc tccgagccgg gtcccgtctc ggacacccag gtctcgtccc 11820 gcagatcggc gagcgagacc tcccgccggg cggccagcgg atggtcccgg ggcaggatca 11880 cccacagcgg gtcgtccagc acctcacagg tgcgcacgga ccgctccagg ctgtgccggg 11940 gggactggag gctccaggtg taggccgcgt ccacctggta gcccgccagt tgggcggcga 12000 cctggtgcgg ggcctcgtgc cggaccgaca gcagcaggtc cagcgaggcc gccgcgtcct 12060 ccaccacctc gtcgagcagg ggttccgtgg agaccagcga cagcacctcc ggggcgtcca 12120 cggcctcgga gccatggccg aagatatgcg tccgcgcggc caggtcgacc tggtggaaga 12180 accgccgccc ggcgacgagg atgcgggagc ccgcggtggt cagccgggcc gtgtggcggc 12240 tgcgcagggt cagcgggagg ccgacgatcc ggtccagccg gtcgagtctg cgctccacgg 12300 tgccgtgccg gacacccgtc cgccgggcca cttccatgag gtctccgcag tgtcccaccg 12360 cgtccagtaa agacagatcg catcggctga caccagcaga cgtcggttct gacccgagag 12420 acaatgtcgg ttcccttttc cgtcaaggac tgtaccgctg aattgtccga agtggctctt 12480 gaattgcttc ggaatcgatc ctaggcagcg ccgctcttcg gattctcctc gccgggaagc 12540 ggaacgcgcc cggccggatg gcgggcgcgc tccgggcgcc gtcccgggaa cgggggacgg 12600 ggcacggcac ggccggccac ccggtccggg cgcgcggcgt ggacctggtc ggcggacggg 12660 tgtcagacct ggtcggtggg gcgtatgaag atctcgtgga cggtcgcgtg gtgcggcgcg 12720 gtcacggcgt agcggaccgc ctccgcgatg tcctgggcct ggagcttgcg gatctggctg 12780 atccgctgct cgtacatctc cttggtggcg gtgtgggtga tgtggccgcg cagctccgtg 12840 tcggtggtgc ccggctcgat gacgacgacc cgcaccccgc gctcggtgac ctcctggcgc 12900 agcgtctcgc tgaacgcgtt cacaccgaac ttcgtggcct ggtagacggc cgcgttgcgg 12960 acgttcaccc ggcccgcgat cgaggacatc tgcaccacgg tgcccttgct gcgcagcaga 13020 tggggaaggg ccgcccgggt catgtacatc aggcccagga gattggtgtc gatcatccgg 13080 gtccagtcgg tggtgtcggc gtcctccacc gggccgagca gcatgatccc ggcgttgttg 13140 acgaggatgt cgaggccgcc cagcgcctcg acggtggagg cgacggcggc gtccaccccc 13200 tgccggtcgg cgacgtcgag ttcgaggaca tggaccttcg ccccggcggc ggtcagctcg 13260 tcacccaggg cgcgcagctt ctcgacccgg cgcgcggcga tggccacggc ggcgccctcg 13320 gcggccaggg cgcgggccgt ggcctcgccg atgcccgagc tcgcgcccgt gatgagcgcg 13380 actttcccct ggagtgcgga tggcatcatt tcctccacat ggtgctgcga tcgtggtgag 13440 cgtatgaaga aggggtgaga cctgccgtgc cggggcgggt tccgtacgcc ggaccgttgc 13500 ggtgggcacg gccgaccggg tacggatggc cgcagttccc cggggagttc ccggggaatg 13560 gtgaataccg cggcgctctc cgatggtctt cggaggacac ccggggattc accgggaatc 13620 agcggccgga gttctccccg tccacggcag acgctatcag cgtcgcattc cccggtgaat 13680 tcccttcggt ggaccgggtt atgactgttt ccgccgggtt atgcgcgccg ccccggcgga 13740 ccggccaccc gcccgggggc tgcggcagat tgggcgccac gacatggcgc gagcagcgat 13800 cggcggtgga tgatgaacga ggcagcgcct cagtccgacc aggtggcacc ggcgtatccg 13860 atgcaccggg tctgcccggt cgacccgccg ccgcaactgg ccgggctgcg gtcccagaag 13920 gccgcgagcc gggtgacgct gtgggacggc agccaggtgt ggctggtgac ctcgcacgcc 13980 ggggcccggg ccgtcctggg cgaccgccgc ttcaccgcgg tgacgagcgc gcccggcttc 14040 ccgatgctga cccgcacctc ccaactggtg cgcgccaacc cggagtcggc gtcgttcatc 14100 cgcatggacg acccgcagca ctcccggctg cgctcgatgc tcacccggga cttcctggcc 14160 cgccgcgccg aggcgctgcg ccccgcggtg cgggagctgc tggacgagat cctgggcggg 14220 ctggtgaagg gggagcggcc ggtcgacctg gtcgccggac tgacgatccc ggtgccctcg 14280 cgggtcatca ccctgctctt cggcgccggt gacgaccgcc gggagttcat cgaggaccgc 14340 agcgcggtcc tcatcgaccg cggctacacc ccggagcagg tcgccaaggc ccgggacgaa 14400 ctcgacggct atctgcggga gctggtcgag gagcggatcg agaacccggg caccgacctg 14460 atcagccggc tcgtcatcga ccaggtgcgg ccggggcatc tgcgggtcga ggagatggtc 14520 ccgatgtgcc ggctgctgct ggtggccggt cacggcacca ccaccagcca ggcgagcctg 14580 agcctgctca gcctgctcac cgacccggag ctggccgggc gcctcaccga ggacccggcc 14640 ctgctgccca aggcggtcga ggagctgctg cgcttccact ccatcgtgca gaacgggctg 14700 gcccgtgccg cggtggagga cgtccagctc gacgatgtgc tcatccgggc gggcgagggc 14760 gtggtgctgt cgctgtcggc gggcaaccgg gacgagacgg tcttccccga cccggaccgg 14820 gtggacgtgg accgcgacgc ccgccgccat ctcgccttcg gccacggcat gcaccagtgc 14880 ctgggccagt ggctggcccg ggtggagctg gaggagatcc tcgccgcggt gctgcgctgg 14940 atgcccggtg cccggctcgc ggtgcccttc gaggagctgg acttccgtca tgaggtgtcc 15000 agttacggcc tcggcgccct cccggtgacc tggtgagcgg cgtggagcgg ctgaccgtcg 15060 tcctcgacgc gtcggcctgc tgcgcgatgg ggcgctgcgc ggccacggcc cccgagatct 15120 2 24 DNA Artificial Sequence Description of Artificial SequencePRIMER 2 tacgcccaga tccccacctt catg 24 3 552 PRT Streptomyces clavuligerus 3 Met Thr His Ser Asp Asn Tyr Gly Asp Asp Pro Pro Gln Gly Arg Arg 1 5 10 15 Arg Ser Arg Gly Arg Ala Ala Thr Ala Val Val Ala Gly Leu Ala Val 20 25 30 Thr Val Gly Leu Gly Tyr Trp Gly Tyr Thr Ser Leu Val Ala Asp Glu 35 40 45 Lys Asp Ser Gly Asp Pro Glu Val Glu Ala Ala Ala Gly Gln Phe Asp 50 55 60 Thr Phe Leu Gly Ala Trp Glu Lys Gly Asp Ala Pro Thr Ala Ala Gly 65 70 75 80 Leu Thr Asp Thr Pro Asp Asn Ala Glu Ser Leu Ile Lys Ser Val Met 85 90 95 Thr Asn Leu Lys Pro Thr Lys Thr Glu Ile Thr Ala Lys Thr Gly Glu 100 105 110 Lys Asn Pro Glu Gly Glu Val Glu Ile Pro Phe Thr Val Arg Met Thr 115 120 125 Leu Pro Gly Ala Gly Glu Tyr Ala Trp Asp Ser Thr Ala Lys Val Val 130 135 140 Gly Gly Gly Lys Glu Trp Lys Val Ala Phe Asn Thr Glu Met Ile His 145 150 155 160 Pro Gln Met Val Pro Gly Gln Thr Leu Ala Leu Lys Ser Arg Glu Arg 165 170 175 Ala Asp Ile Leu Asp Ala Asn Gly Asn Val Leu Gln Ala Ala Ser Ile 180 185 190 Ile Gly Ala Val Asp Pro Arg Thr Gly Lys Gly Ser Ala Gly Leu Gln 195 200 205 Ser Arg Tyr Asp Lys Gln Leu Thr Gly Gly Ser Gly Ala Ala Arg Ser 210 215 220 Val Val Ile Leu Asp Arg Glu Ser Gly Gln Val Val Lys Lys Leu Thr 225 230 235 240 Gly Leu Lys Asp Thr Glu Gly Lys Pro Val Lys Thr Thr Ile Asp Pro 245 250 255 Arg Val Gln Ser Ala Ala Ala Ala Ala Leu Glu Gly Ser Lys Lys Asn 260 265 270 Ala Ala Ile Val Ala Val Asp Pro Ala Thr Gly Asn Ile Leu Ala Ala 275 280 285 Ala Asn Val Pro Ser Gly Met Asn Arg Ala Leu Glu Gly Arg Tyr Pro 290 295 300 Pro Gly Ser Thr Phe Lys Val Val Thr Thr Ala Ala Leu Leu Gln Gln 305 310 315 320 Gly Met Asn Pro Glu Glu Arg Ala Asp Cys Pro Lys Phe Ala His Val 325 330 335 Asn Gly Gln Ser Phe Glu Asn Gln Asp Gln Phe Thr Leu Pro Ala Gly 340 345 350 Ser Thr Phe Arg Asp Ser Phe Ala His Ser Cys Asn Thr Phe Phe Val 355 360 365 Asn Ser Arg Ser Lys Leu Ser Glu Ser Ser Leu Lys Gln Ala Ala Glu 370 375 380 Ala Phe Gly Ile Gly Gly Thr Trp Asp Val Gly Ala Ser Thr Phe Asp 385 390 395 400 Gly Ser Val Pro Val Ser Asn Ser Glu Asn Asp Lys Ala Ala Ser Thr 405 410 415 Ile Gly Gln Ala Arg Val Glu Ala Ser Pro Leu Val Met Ala Ser Ile 420 425 430 Ala Ala Thr Val Lys Gln Gly Glu Phe Lys Gln Pro Val Leu Val Pro 435 440 445 Asp Ala Val Lys Lys Pro His Gln Ala Pro Arg Met Ala Pro Gly Ile 450 455 460 Val Asp Ser Leu Arg Ser Met Met Arg Ser Thr Val Thr Asp Gly Ala 465 470 475 480 Gly Asp Ala Leu Arg Gly Leu Gly Gly Gln Pro His Ala Lys Thr Gly 485 490 495 Thr Ala Glu Phe Gly Thr Glu Lys Pro Pro Lys Thr His Ala Trp Met 500 505 510 Ile Gly Tyr Gln Gly Asp Arg Asn Ile Ala Trp Ser Val Leu Leu Glu 515 520 525 Asp Gly Gly Ser Gly Gly Ala Asp Ala Gly Pro Val Ala Ala Lys Phe 530 535 540 Leu Ser Asn Leu Ala Ala Gly Glx 545 550 4 574 PRT Streptomyces clavuligerus 4 Met Ser Arg Val Ser Thr Ala Pro Ser Gly Lys Pro Thr Ala Ala His 1 5 10 15 Ala Leu Leu Ser Arg Leu Arg Asp His Gly Val Gly Lys Val Phe Gly 20 25 30 Val Val Gly Arg Glu Ala Ala Ser Ile Leu Phe Asp Glu Val Glu Gly 35 40 45 Ile Asp Phe Val Leu Thr Arg His Glu Phe Thr Ala Gly Val Ala Ala 50 55 60 Asp Val Leu Ala Arg Ile Thr Gly Arg Pro Gln Ala Cys Trp Ala Thr 65 70 75 80 Leu Gly Pro Gly Met Thr Asn Leu Ser Thr Gly Ile Ala Thr Ser Val 85 90 95 Leu Asp Arg Ser Pro Val Ile Ala Leu Ala Ala Gln Ser Glu Ser His 100 105 110 Asp Ile Phe Pro Asn Asp Thr His Gln Cys Leu Asp Ser Val Ala Ile 115 120 125 Val Ala Pro Met Ser Lys Tyr Ala Val Glu Leu Gln Arg Pro His Glu 130 135 140 Ile Thr Asp Leu Val Asp Ser Ala Val Asn Ala Ala Met Thr Glu Pro 145 150 155 160 Val Gly Pro Ser Phe Ile Ser Leu Pro Val Asp Leu Leu Gly Ser Ser 165 170 175 Glu Gly Ile Asp Thr Thr Val Pro Asn Pro Pro Ala Asn Thr Pro Ala 180 185 190 Lys Pro Val Gly Val Val Ala Asp Gly Trp Gln Lys Ala Ala Asp Gln 195 200 205 Ala Ala Ala Leu Leu Ala Glu Ala Lys His Pro Val Leu Val Val Gly 210 215 220 Ala Ala Ala Ile Arg Ser Gly Ala Val Pro Ala Ile Arg Ala Leu Ala 225 230 235 240 Glu Arg Leu Asn Ile Pro Val Ile Thr Thr Tyr Ile Ala Lys Gly Val 245 250 255 Leu Pro Val Gly His Glu Leu Asn Tyr Gly Ala Val Thr Gly Tyr Met 260 265 270 Asp Gly Ile Leu Asn Phe Pro Ala Leu Gln Thr Met Phe Ala Pro Val 275 280 285 Asp Leu Val Leu Thr Val Gly Tyr Asp Tyr Ala Glu Asp Leu Arg Pro 290 295 300 Ser Met Trp Gln Lys Gly Ile Glu Lys Lys Thr Val Arg Ile Ser Pro 305 310 315 320 Thr Val Asn Pro Ile Pro Arg Val Tyr Arg Pro Asp Val Asp Val Val 325 330 335 Thr Asp Val Leu Ala Phe Val Glu His Phe Glu Thr Ala Thr Ala Ser 340 345 350 Phe Gly Ala Lys Gln Arg His Asp Ile Glu Pro Leu Arg Ala Arg Ile 355 360 365 Ala Glu Phe Leu Ala Asp Pro Glu Thr Tyr Glu Asp Gly Met Arg Val 370 375 380 His Gln Val Ile Asp Ser Met Asn Thr Val Met Glu Glu Ala Ala Glu 385 390 395 400 Pro Gly Glu Gly Thr Ile Val Ser Asp Ile Gly Phe Phe Arg His Tyr 405 410 415 Gly Val Leu Phe Ala Arg Ala Asp Gln Pro Phe Gly Phe Leu Thr Ser 420 425 430 Ala Gly Cys Ser Ser Phe Gly Tyr Gly Ile Pro Ala Ala Ile Gly Ala 435 440 445 Gln Met Ala Arg Pro Asp Gln Pro Thr Phe Leu Ile Ala Gly Asp Gly 450 455 460 Gly Phe His Ser Asn Ser Ser Asp Leu Glu Thr Ile Ala Arg Leu Asn 465 470 475 480 Leu Pro Ile Val Thr Val Val Val Asn Asn Asp Thr Asn Gly Leu Ile 485 490 495 Glu Leu Tyr Gln Asn Ile Gly His His Arg Ser His Asp Pro Ala Val 500 505 510 Lys Phe Gly Gly Val Asp Phe Val Ala Leu Ala Glu Ala Asn Gly Val 515 520 525 Asp Ala Thr Arg Ala Thr Asn Arg Glu Glu Leu Leu Ala Ala Leu Arg 530 535 540 Lys Gly Ala Glu Leu Gly Arg Pro Phe Leu Ile Glu Val Pro Val Asn 545 550 555 560 Tyr Asp Phe Gln Pro Gly Gly Phe Gly Ala Leu Ser Ile Glx 565 570 5 514 PRT Streptomyces clavuligerus 5 Met Gly Ala Pro Val Leu Pro Ala Ala Phe Gly Phe Leu Ala Ser Ala 1 5 10 15 Arg Thr Gly Gly Gly Arg Ala Pro Gly Pro Val Phe Ala Thr Arg Gly 20 25 30 Ser His Thr Asp Ile Asp Thr Pro Gln Gly Glu Arg Ser Leu Ala Ala 35 40 45 Thr Leu Val His Ala Pro Ser Val Ala Pro Asp Arg Ala Val Ala Arg 50 55 60 Ser Leu Thr Gly Ala Pro Thr Thr Ala Val Leu Ala Gly Glu Ile Tyr 65 70 75 80 Asn Arg Asp Glu Leu Leu Ser Val Leu Pro Ala Gly Pro Ala Pro Glu 85 90 95 Gly Asp Ala Glu Leu Val Leu Arg Leu Leu Glu Arg Tyr Asp Leu His 100 105 110 Ala Phe Arg Leu Val Asn Gly Arg Phe Ala Thr Val Val Arg Thr Gly 115 120 125 Asp Arg Val Leu Leu Ala Thr Asp His Ala Gly Ser Val Pro Leu Tyr 130 135 140 Thr Cys Val Ala Pro Gly Glu Val Arg Ala Ser Thr Glu Ala Lys Ala 145 150 155 160 Leu Ala Ala His Arg Asp Pro Lys Gly Phe Pro Leu Ala Asp Ala Arg 165 170 175 Arg Val Ala Gly Leu Thr Gly Val Tyr Gln Val Pro Ala Gly Ala Val 180 185 190 Met Asp Ile Asp Leu Gly Ser Gly Thr Ala Val Thr His Arg Thr Trp 195 200 205 Thr Pro Gly Leu Ser Arg Arg Ile Leu Pro Glu Gly Glu Ala Val Ala 210 215 220 Ala Val Arg Ala Ala Leu Glu Lys Ala Val Ala Gln Arg Val Thr Pro 225 230 235 240 Gly Asp Thr Pro Leu Val Val Leu Ser Gly Gly Ile Asp Ser Ser Gly 245 250 255 Val Ala Ala Cys Ala His Arg Ala Ala Gly Glu Leu Asp Thr Val Ser 260 265 270 Met Gly Thr Asp Thr Ser Asn Glu Phe Arg Glu Ala Arg Ala Val Val 275 280 285 Asp His Leu Arg Thr Arg His Arg Glu Ile Thr Ile Pro Thr Thr Glu 290 295 300 Leu Leu Ala Gln Leu Pro Tyr Ala Val Trp Ala Ser Glu Ser Val Asp 305 310 315 320 Pro Asp Ile Ile Glu Tyr Leu Leu Pro Leu Thr Ala Leu Tyr Arg Ala 325 330 335 Leu Asp Gly Pro Glu Arg Arg Ile Leu Thr Gly Tyr Gly Ala Asp Ile 340 345 350 Pro Leu Gly Gly Met His Arg Glu Asp Arg Leu Pro Ala Leu Asp Thr 355 360 365 Val Leu Ala His Asp Met Ala Thr Phe Asp Gly Leu Asn Glu Met Ser 370 375 380 Pro Val Leu Ser Thr Leu Ala Gly His Trp Thr Thr His Pro Tyr Trp 385 390 395 400 Asp Arg Glu Val Leu Asp Leu Leu Val Ser Leu Glu Ala Gly Leu Lys 405 410 415 Arg Arg His Gly Arg Asp Lys Trp Val Leu Arg Ala Ala Met Ala Asp 420 425 430 Ala Leu Pro Ala Glu Thr Val Asn Arg Pro Lys Leu Gly Val His Glu 435 440 445 Gly Ser Gly Thr Thr Ser Ser Phe Ser Arg Leu Leu Leu Asp His Gly 450 455 460 Val Ala Glu Asp Arg Val His Glu Ala Lys Arg Gln Val Val Arg Glu 465 470 475 480 Leu Phe Asp Leu Thr Val Gly Gly Gly Arg His Pro Ser Glu Val Asp 485 490 495 Thr Asp Asp Val Val Arg Ser Val Ala Asp Arg Thr Ala Arg Gly Ala 500 505 510 Ala Glx 6 314 PRT Streptomyces clavuligerus 6 Val Glu Arg Ile Asp Ser His Val Ser Pro Arg Tyr Ala Gln Ile Pro 1 5 10 15 Thr Phe Met Arg Leu Pro His Asp Pro Gln Pro Arg Gly Tyr Asp Val 20 25 30 Val Val Ile Gly Ala Pro Tyr Asp Gly Gly Thr Ser Tyr Arg Pro Gly 35 40 45 Ala Arg Phe Gly Pro Gln Ala Ile Arg Ser Glu Ser Gly Leu Ile His 50 55 60 Gly Val Gly Ile Asp Arg Gly Pro Gly Thr Phe Asp Leu Ile Asn Cys 65 70 75 80 Val Asp Ala Gly Asp Ile Asn Leu Thr Pro Phe Asp Met Asn Ile Ala 85 90 95 Ile Asp Thr Ala Gln Ser His Leu Ser Gly Leu Leu Lys Ala Asn Ala 100 105 110 Ala Phe Leu Met Ile Gly Gly Asp His Ser Leu Thr Val Ala Ala Leu 115 120 125 Arg Ala Val Ala Glu Gln His Gly Pro Leu Ala Val Val His Leu Asp 130 135 140 Ala His Ser Asp Thr Asn Pro Ala Phe Tyr Gly Gly Arg Tyr His His 145 150 155 160 Gly Thr Pro Phe Arg His Gly Ile Asp Glu Lys Leu Ile Asp Pro Ala 165 170 175 Ala Met Val Gln Ile Gly Ile Arg Gly His Asn Pro Lys Pro Asp Ser 180 185 190 Leu Asp Tyr Ala Arg Gly His Gly Val Arg Val Val Thr Ala Asp Glu 195 200 205 Phe Gly Glu Leu Gly Val Gly Gly Thr Ala Asp Leu Ile Arg Glu Lys 210 215 220 Val Gly Gln Arg Pro Val Tyr Val Ser Val Asp Ile Asp Val Val Asp 225 230 235 240 Pro Ala Phe Ala Pro Gly Thr Gly Thr Pro Ala Pro Gly Gly Leu Leu 245 250 255 Ser Arg Glu Val Leu Ala Leu Leu Arg Cys Val Gly Asp Leu Lys Pro 260 265 270 Val Gly Phe Asp Val Met Glu Val Ser Pro Leu Tyr Asp His Gly Gly 275 280 285 Ile Thr Ser Ile Leu Ala Thr Glu Ile Gly Ala Glu Leu Leu Tyr Gln 290 295 300 Tyr Ala Arg Ala His Arg Thr Gln Leu Glx 305 310 7 326 PRT Streptomyces clavuligerus 7 Met Ala Ser Pro Ile Val Asp Cys Thr Pro Tyr Arg Asp Glu Leu Leu 1 5 10 15 Ala Leu Ala Ser Glu Leu Pro Glu Val Pro Arg Ala Asp Leu His Gly 20 25 30 Phe Leu Asp Glu Ala Lys Thr Leu Ala Ala Arg Leu Pro Glu Gly Leu 35 40 45 Ala Ala Ala Leu Asp Thr Phe Asn Ala Val Gly Ser Glu Asp Gly Tyr 50 55 60 Leu Leu Leu Arg Gly Leu Pro Val Asp Asp Ser Glu Leu Pro Glu Thr 65 70 75 80 Pro Thr Ser Thr Pro Ala Pro Leu Asp Arg Lys Arg Leu Val Met Glu 85 90 95 Ala Met Leu Ala Leu Ala Gly Arg Arg Leu Gly Leu His Thr Gly Tyr 100 105 110 Gln Glu Leu Arg Ser Gly Thr Val Tyr His Asp Val Tyr Pro Ser Pro 115 120 125 Gly Ala His Tyr Leu Ser Ser Glu Thr Ser Glu Thr Leu Leu Glu Phe 130 135 140 His Thr Glu Met Ala Tyr His Ile Leu Gln Pro Asn Tyr Val Met Leu 145 150 155 160 Ala Cys Ser Arg Ala Asp His Glu Asn Arg Ala Glu Thr Leu Val Gly 165 170 175 Ser Val Arg Lys Ala Leu Pro Leu Leu Asp Glu Lys Thr Arg Ala Arg 180 185 190 Leu Phe Asp Arg Lys Val Pro Cys Cys Val Asp Val Ala Phe Arg Gly 195 200 205 Gly Val Asp Asp Pro Gly Ala Ile Ala Asn Val Lys Pro Leu Tyr Gly 210 215 220 Asp Ala Asn Asp Pro Phe Leu Gly Tyr Asp Arg Glu Leu Leu Ala Pro 225 230 235 240 Glu Asp Pro Ala Asp Lys Glu Ala Val Ala His Leu Ser Gln Ala Leu 245 250 255 Asp Asp Val Thr Val Gly Val Lys Leu Val Pro Gly Asp Val Leu Ile 260 265 270 Ile Asp Asn Phe Arg Thr Thr His Ala Arg Thr Pro Phe Ser Pro Arg 275 280 285 Trp Asp Gly Lys Asp Arg Trp Leu His Arg Val Tyr Ile Arg Thr Asp 290 295 300 Arg Asn Gly Gln Leu Ser Gly Gly Glu Arg Ala Gly Asp Thr Ile Ser 305 310 315 320 Phe Ser Pro Arg Arg Glx 325 8 394 PRT Streptomyces clavuligerus 8 Met Ser Asp Ser Thr Pro Lys Thr Pro Arg Gly Phe Val Val His Thr 1 5 10 15 Ala Pro Val Gly Leu Ala Asp Asp Gly Arg Asp Asp Phe Thr Val Leu 20 25 30 Ala Ser Thr Ala Pro Ala Thr Val Ser Ala Val Phe Thr Arg Ser Arg 35 40 45 Phe Ala Gly Pro Ser Val Val Leu Cys Arg Glu Ala Val Ala Asp Gly 50 55 60 Gln Ala Arg Gly Val Val Val Leu Ala Arg Asn Ala Asn Val Ala Thr 65 70 75 80 Gly Leu Glu Gly Glu Glu Asn Ala Arg Glu Val Arg Glu Ala Val Ala 85 90 95 Arg Ala Leu Gly Leu Pro Glu Gly Glu Met Leu Ile Ala Ser Thr Gly 100 105 110 Val Ile Gly Arg Gln Tyr Pro Met Glu Ser Ile Arg Glu His Leu Lys 115 120 125 Thr Leu Glu Trp Pro Ala Gly Glu Gly Gly Phe Asp Arg Ala Ala Arg 130 135 140 Ala Ile Met Thr Thr Asp Thr Arg Pro Lys Glu Val Arg Val Ser Val 145 150 155 160 Gly Gly Ala Thr Leu Val Gly Ile Ala Lys Gly Val Gly Met Leu Glu 165 170 175 Pro Asp Met Ala Thr Leu Leu Thr Phe Phe Ala Thr Asp Ala Arg Leu 180 185 190 Asp Pro Ala Glu Gln Asp Arg Leu Phe Arg Arg Val Met Asp Arg Thr 195 200 205 Phe Asn Ala Val Ser Ile Asp Thr Asp Thr Ser Thr Ser Asp Thr Ala 210 215 220 Val Leu Phe Ala Asn Gly Leu Ala Gly Glu Val Asp Ala Gly Glu Phe 225 230 235 240 Glu Glu Ala Leu His Thr Ala Ala Leu Ala Leu Val Lys Asp Ile Ala 245 250 255 Ser Asp Gly Glu Gly Ala Ala Lys Leu Ile Glu Val Gln Val Thr Gly 260 265 270 Ala Arg Asp Asp Ala Gln Ala Lys Arg Val Gly Lys Thr Val Val Asn 275 280 285 Ser Pro Leu Val Lys Thr Ala Val His Gly Cys Asp Pro Asn Trp Gly 290 295 300 Arg Val Ala Met Ala Ile Gly Lys Cys Ser Asp Asp Thr Asp Ile Asp 305 310 315 320 Gln Glu Arg Val Thr Ile Arg Phe Gly Glu Val Glu Val Tyr Pro Pro 325 330 335 Lys Ala Arg Gly Asp Gln Ala Asp Asp Ala Leu Arg Ala Ala Val Ala 340 345 350 Glu His Leu Arg Gly Asp Glu Val Val Ile Gly Ile Asp Leu Ala Ile 355 360 365 Ala Asp Gly Ala Phe Thr Val Tyr Gly Cys Asp Leu Thr Glu Gly Tyr 370 375 380 Val Arg Leu Asn Ser Glu Tyr Thr Thr Glx 385 390 9 556 PRT Streptomyces clavuligerus 9 Met Glu Thr Thr Arg Ser Thr Thr Ala Asp Glu Gly Phe Asp Ala Gly 1 5 10 15 Val Arg Gly Val Val Ala Pro Thr Asp Ala Pro Gly Gly Thr Leu Arg 20 25 30 Leu Val Arg Thr Asp Asp Phe Asp Ser Leu Asp Pro Gly Asn Thr Tyr 35 40 45 Tyr Ala Tyr Thr Trp Asn Phe Leu Arg Leu Ile Gly Arg Thr Leu Val 50 55 60 Thr Phe Asp Thr Ala Pro Gly Lys Ala Gly Gln Arg Leu Val Pro Asp 65 70 75 80 Leu Ala Glu Ser Leu Gly Glu Ser Ser Glu Asp Gly Arg Val Trp Thr 85 90 95 Tyr Arg Leu Arg Glu Gly Leu Arg Tyr Glu Asp Gly Thr Pro Val Val 100 105 110 Ser Ala Asp Ile Lys His Ala Ile Ala Arg Ser Asn Tyr Gly Thr Asp 115 120 125 Val Leu Gly Ala Gly Pro Thr Tyr Phe Arg His Leu Leu Gly Thr Glu 130 135 140 Tyr Gly Gly Pro Trp Arg Glu Pro Asp Ala Asp Gly Pro Val Thr Leu 145 150 155 160 Glu Thr Pro Asp Glu Arg Thr Leu Val Phe Arg Leu Arg Glu Pro Phe 165 170 175 Ala Gly Met Asp Leu Leu Ala Thr Met Pro Ser Thr Thr Pro Val Pro 180 185 190 Arg Asp Arg Asp Thr Gly Ala Glu Tyr Arg Leu Arg Pro Val Ala Thr 195 200 205 Gly Pro Tyr Arg Ile Val Ser Tyr Thr Arg Gly Glu Leu Ala Val Leu 210 215 220 Glu Pro Asn Pro His Trp Asp Pro Glu Thr Asp Pro Val Arg Val Gln 225 230 235 240 Arg Ala Ser Arg Ile Glu Val His Leu Gly Lys Asp Pro His Glu Val 245 250 255 Asp Arg Met Leu Leu Ala Gly Glu Ala His Val Asp Leu Ala Gly Phe 260 265 270 Gly Val Gln Pro Ala Ala Gln Glu Arg Ile Leu Ala Glu Pro Glu Leu 275 280 285 Arg Ala His Ala Asp Asn Pro Leu Thr Gly Phe Thr Trp Ile Tyr Cys 290 295 300 Leu Ser Ser Arg Ile Ala Pro Phe Asp Asn Val His Cys Arg Arg Ala 305 310 315 320 Val Gln Phe Ala Thr Asp Lys Ala Ala Met Gln Glu Ala Tyr Gly Gly 325 330 335 Ala Val Gly Gly Asp Ile Ala Thr Thr Leu Leu Pro Pro Thr Leu Asp 340 345 350 Gly Tyr Lys His Phe Asp Arg Tyr Pro Val Gly Pro Glu Gly Thr Gly 355 360 365 Asp Leu Glu Ala Ala Arg Ala Glu Leu Lys Leu Ala Gly Met Pro Asp 370 375 380 Gly Phe Arg Thr Arg Ile Ala Ala Arg Lys Asp Arg Leu Lys Glu Tyr 385 390 395 400 Arg Ala Ala Glu Ala Leu Ala Ala Gly Leu Ala Arg Val Gly Ile Glu 405 410 415 Ala Glu Val Leu Asp Phe Pro Ser Gly Asp Tyr Phe Asp Arg Tyr Gly 420 425 430 Gly Cys Pro Glu Tyr Leu Arg Glu His Gly Ile Gly Ile Ile Met Phe 435 440 445 Gly Trp Gly Ala Asp Phe Pro Asp Gly Tyr Gly Phe Leu Gln Gln Ile 450 455 460 Thr Asp Gly Arg Ala Ile Lys Glu Arg Gly Asn Gln Asn Met Gly Glu 465 470 475 480 Leu Asp Asp Pro Glu Ile Asn Ala Leu Leu Asp Glu Gly Ala Gln Cys 485 490 495 Ala Asp Pro Ala Arg Arg Ala Glu Ile Trp His Arg Ile Asp Gln Leu 500 505 510 Thr Met Asp His Ala Val Ile Val Pro Tyr Leu Tyr Pro Arg Ser Leu 515 520 525 Leu Tyr Arg His Pro Asp Thr Arg Asn Ala Phe Val Thr Gly Ser Phe 530 535 540 Gly Met Tyr Asp Tyr Val Ala Leu Gly Ala Lys Glx 545 550 555 10 433 PRT Streptomyces clavuligerus 10 Met Glu Val Ala Arg Arg Thr Gly Val Arg His Gly Thr Val Glu Arg 1 5 10 15 Arg Leu Asp Arg Leu Asp Arg Ile Val Gly Leu Pro Leu Thr Leu Arg 20 25 30 Ser Arg His Thr Ala Arg Leu Thr Thr Ala Gly Ser Arg Ile Leu Val 35 40 45 Ala Gly Arg Arg Phe Phe His Gln Val Asp Leu Ala Ala Arg Thr His 50 55 60 Ile Phe Gly His Gly Ser Glu Ala Val Asp Ala Pro Glu Val Leu Ser 65 70 75 80 Leu Val Ser Thr Glu Pro Leu Leu Asp Glu Val Val Glu Asp Ala Ala 85 90 95 Ala Ser Leu Asp Leu Leu Leu Ser Val Arg His Glu Ala Pro His Gln 100 105 110 Val Ala Ala Gln Leu Ala Gly Tyr Gln Val Asp Ala Ala Tyr Thr Trp 115 120 125 Ser Leu Gln Ser Pro Arg His Ser Leu Glu Arg Ser Val Arg Thr Cys 130 135 140 Glu Val Leu Asp Asp Pro Leu Trp Val Ile Leu Pro Arg Asp His Pro 145 150 155 160 Leu Ala Ala Arg Arg Glu Val Ser Leu Ala Asp Leu Arg Asp Glu Thr 165 170 175 Trp Val Ser Glu Thr Gly Pro Gly Ser Glu Ile Leu Val Thr Arg Val 180 185 190 Phe Gln Leu Ala Gly Leu Thr Ala Pro Thr Arg Leu His Ile Thr Gly 195 200 205 Ala Ser Val Ala Arg Gly Ile Leu Arg Arg Gly Asp Ala Ile Gly Leu 210 215 220 Gly Ser Pro Thr His Pro Ala Val Gln Asp Pro Ser Leu Val Arg Arg 225 230 235 240 Ser Leu Ala Glu Arg Pro Arg Arg Thr Thr Ser Leu Leu Val Asp Pro 245 250 255 Thr Ile Val Pro Arg Ala Leu Ala Gly Arg Leu Ala Leu Leu Ile Ala 260 265 270 Glu Val Gln Leu Arg Arg Phe Ala Glu His His Arg Asp Leu Leu Asp 275 280 285 Glu Pro Trp Trp Ala Gln Trp Tyr Ala Glu Arg Thr Gly Ala Asp Ala 290 295 300 Arg Arg Phe Gly Ala Gly Pro Asp Gln Gly Ser Val Pro Gly Gln Ala 305 310 315 320 Glu Gly Arg Lys Leu Asp Val Asp Asp Leu His Leu Leu Gln Ala Val 325 330 335 Ala Arg His Gly Ser Ile Asn Arg Ala Ala Ala Val Leu Ser Ile Ser 340 345 350 Gln Ser Ala Leu Thr Arg Arg Ile His Arg Leu Glu Gln Ser Leu Gly 355 360 365 Ala Arg Leu Leu Leu Arg Ser Pro Arg Gly Thr Ser Leu Thr Gly Pro 370 375 380 Thr Arg Gln Phe Leu Arg Gln Leu Ala Leu Tyr Glu Ala Glu Phe Arg 385 390 395 400 Glu Ala Ala Leu Ala Cys Arg Ser Val Glu Arg Pro Leu Ala Gln Gly 405 410 415 His Trp Pro Ile Arg Arg Gly Val Ala Ala Gly Ala Arg Met Ser Gly 420 425 430 Glx 11 248 PRT Streptomyces clavuligerus 11 Met Pro Ser Ala Leu Gln Gly Lys Val Ala Leu Ile Thr Gly Ala Ser 1 5 10 15 Ser Gly Ile Gly Glu Ala Thr Ala Arg Ala Leu Ala Ala Glu Gly Ala 20 25 30 Ala Val Ala Ile Ala Ala Arg Arg Val Glu Lys Leu Arg Ala Leu Gly 35 40 45 Asp Glu Leu Thr Ala Ala Gly Ala Lys Val His Val Leu Glu Leu Asp 50 55 60 Val Ala Asp Arg Gln Gly Val Asp Ala Ala Val Ala Ser Thr Val Glu 65 70 75 80 Ala Leu Gly Gly Leu Asp Ile Leu Val Asn Asn Ala Gly Ile Met Leu 85 90 95 Leu Gly Pro Val Glu Asp Ala Asp Thr Thr Asp Trp Thr Arg Met Ile 100 105 110 Asp Thr Asn Leu Leu Gly Leu Met Tyr Met Thr Arg Ala Ala Leu Pro 115 120 125 His Leu Leu Arg Ser Lys Gly Thr Val Val Gln Met Ser Ser Ile Ala 130 135 140 Gly Arg Val Asn Val Arg Asn Ala Ala Val Tyr Gln Ala Thr Lys Phe 145 150 155 160 Gly Val Asn Ala Phe Ser Glu Thr Leu Arg Gln Glu Val Thr Glu Arg 165 170 175 Gly Val Arg Val Val Val Ile Glu Pro Gly Thr Thr Asp Thr Glu Leu 180 185 190 Arg Gly His Ile Thr His Thr Ala Thr Lys Glu Met Tyr Glu Gln Arg 195 200 205 Ile Ser Gln Ile Arg Lys Leu Gln Ala Gln Asp Ile Ala Glu Ala Val 210 215 220 Arg Tyr Ala Val Thr Ala Pro His His Ala Thr Val His Glu Ile Phe 225 230 235 240 Ile Arg Pro Thr Asp Gln Val Glx 245 12 409 PRT Streptomyces clavuligerus 12 Met Met Asn Glu Ala Ala Pro Gln Ser Asp Gln Val Ala Pro Ala Tyr 1 5 10 15 Pro Met His Arg Val Cys Pro Val Asp Pro Pro Pro Gln Leu Ala Gly 20 25 30 Leu Arg Ser Gln Lys Ala Ala Ser Arg Val Thr Leu Trp Asp Gly Ser 35 40 45 Gln Val Trp Leu Val Thr Ser His Ala Gly Ala Arg Ala Val Leu Gly 50 55 60 Asp Arg Arg Phe Thr Ala Val Thr Ser Ala Pro Gly Phe Pro Met Leu 65 70 75 80 Thr Arg Thr Ser Gln Leu Val Arg Ala Asn Pro Glu Ser Ala Ser Phe 85 90 95 Ile Arg Met Asp Asp Pro Gln His Ser Arg Leu Arg Ser Met Leu Thr 100 105 110 Arg Asp Phe Leu Ala Arg Arg Ala Glu Ala Leu Arg Pro Ala Val Arg 115 120 125 Glu Leu Leu Asp Glu Ile Leu Gly Gly Leu Val Lys Gly Glu Arg Pro 130 135 140 Val Asp Leu Val Ala Gly Leu Thr Ile Pro Val Pro Ser Arg Val Ile 145 150 155 160 Thr Leu Leu Phe Gly Ala Gly Asp Asp Arg Arg Glu Phe Ile Glu Asp 165 170 175 Arg Ser Ala Val Leu Ile Asp Arg Gly Tyr Thr Pro Glu Gln Val Ala 180 185 190 Lys Ala Arg Asp Glu Leu Asp Gly Tyr Leu Arg Glu Leu Val Glu Glu 195 200 205 Arg Ile Glu Asn Pro Gly Thr Asp Leu Ile Ser Arg Leu Val Ile Asp 210 215 220 Gln Val Arg Pro Gly His Leu Arg Val Glu Glu Met Val Pro Met Cys 225 230 235 240 Arg Leu Leu Leu Val Ala Gly His Gly Thr Thr Thr Ser Gln Ala Ser 245 250 255 Leu Ser Leu Leu Ser Leu Leu Thr Asp Pro Glu Leu Ala Gly Arg Leu 260 265 270 Thr Glu Asp Pro Ala Leu Leu Pro Lys Ala Val Glu Glu Leu Leu Arg 275 280 285 Phe His Ser Ile Val Gln Asn Gly Leu Ala Arg Ala Ala Val Glu Asp 290 295 300 Val Gln Leu Asp Asp Val Leu Ile Arg Ala Gly Glu Gly Val Val Leu 305 310 315 320 Ser Leu Ser Ala Gly Asn Arg Asp Glu Thr Val Phe Pro Asp Pro Asp 325 330 335 Arg Val Asp Val Asp Arg Asp Ala Arg Arg His Leu Ala Phe Gly His 340 345 350 Gly Met His Gln Cys Leu Gly Gln Trp Leu Ala Arg Val Glu Leu Glu 355 360 365 Glu Ile Leu Ala Ala Val Leu Arg Trp Met Pro Gly Ala Arg Leu Ala 370 375 380 Val Pro Phe Glu Glu Leu Asp Phe Arg His Glu Val Ser Ser Tyr Gly 385 390 395 400 Leu Gly Ala Leu Pro Val Thr Trp Glx 405 13 11604 DNA Streptomyces clavuligerus 13 gaattcgagt catcgggttc ggcgacggat gggcggttcg gccacgcacc gtcactcttc 60 gtcccctctt cacaagaact cccgatacgt ggagaagaga gcgtgaagag cgcgtccggt 120 cagggttgcc gagaaccgtc caccatgacg gagcctggta ctgacggagt cgggagaccg 180 ctcatgtccc gtgtatcgac cgcccccagc ggcaagccta ccgccgctca cgccctcctg 240 tcacggttgc gtgatcacgg tgtggggaag gtgtttgggg ttgtcggccg agaggccgcg 300 tcgattctct tcgacgaggt cgaccccatc gacttcgttc tgacccgcca cgagttcacc 360 gcgggtgtcg ccgctgatgt cctcgcgcgg atcaccggtc gcccccaggc gtgctgggcc 420 accctgggcc ccggtatgac caacctctcc accggtatcg ccacgtccgt cctggaccgc 480 tcgccggtca tcgcgctcgc cgcgcagtcg gagtcgcacg acatcttccc gaacgacacc 540 caccagtgcc tggactcggt ggcgatcgtc gccccgatgt ccttgtacgc cgtggagctc 600 cagcggcccc acgagatcac cgacctcgtc gactccgccg tgaacgcggc catgaccgag 660 ccggtcgggc cctccttcat ctccctcccg gtggacctgc tcggctcctc cgagggcatc 720 gacaccaccg tccccaaccc gccggcgaac accccggcga aaccggtcgg cgtcgtcgcc 780 gacggctggc agaaggccgc cgaccaggcc gccgccctgc tcgccgaggc caagcacccg 840 gtgctcgtcg tcggagcggc cgcgatccgc tcgggcgccg tcccggcgat ccgcgccctg 900 gccgagcgcc tgaacatccc ggtcatcacg acctacatcg ccaagggtgt cctgccggtc 960 ggccacgagc tgaactacgg cgccgtcacc ggctacatgg acggcatcct caacttcccg 1020 gcgctccaga ccatgttcgc cccggtggac ctcgtcctca ccgtcggcta cgactacgcc 1080 gaggacctgc gcccgtccat gtggcagaag ggcatcgaga agaagaccgt ccgtatctcc 1140 ccgacggtca acccgatccc ccgggtctac cggcccgacg tcgacgtcgt caccgacgtc 1200 ctcgccttcg tggagcactt cgagaccgcg accgcctcct tcggggccaa gcagcgccac 1260 gacatcgagc cgctgcgcgc ccggatcgcg gagttcctgg ccgacccgga gacctacgag 1320 gacggcatgc gcgtccacca ggtcatcgac tccatgaaca ccgtcatgga ggaggccgcc 1380 gagcccggcg agggcacgat cgtctccgac atcggcttct tccgtcacta cggtgtgctc 1440 ttcgcccgcg ccgaccagcc cttcggcttc ctcacctcgg cgggctgctc cagcttcggc 1500 tacggcatcc ccgccgccat cggcgcccag atggcccgcc cggaccagcc gaccttcctc 1560 atcgcgggtg acggcggctt ccactccaac agctccgacc tggagaccat cgcccggctc 1620 aacctgccga tcgtgaccgt cgtcgtcaac aacgacacca acggcctgat cgagctgtac 1680 cagaacatcg gtcaccaccg cagccacgac ccggcggtca agttcggcgg cgtcgacttc 1740 gtcgcgctcg ccgaggccaa cggtgtcgac gccacccgcg ccaccaaccg cgaggagctg 1800 ctcgcggccc tgcgcaaggg tgccgagctg ggtcgtccgt tcctcatcga ggtcccggtc 1860 aactacgact tccagccggg cggcttcggc gccctgagca tctgatcatg ggggcaccgg 1920 ttcttccggc tgccttcggg ttcctggcct ccgcccgaac gggcgggggc cgggcccccg 1980 gcccggtctt cgcgacccgg ggcagccaca ccgacatcga cacgccccag ggggagcgct 2040 cgctcgcggc gaccctggtg cacgccccct cggtcgcgcc cgaccgcgcg gtggcgcgct 2100 ccctcaccgg cgcgcccacc accgcggtgc tcgccggtga gatctacaac cgggacgaac 2160 tcctctccgt gctgcccgcc ggacccgcgc cggaggggga cgcggagctg gtcctgcggc 2220 tgctggaacg ctatgacctg catgccttcc ggctggtgaa cgggcgcttc gcgaccgtgg 2280 tgcggaccgg ggaccgggtc ctgctcgcca ccgaccacgc cggttcggtg ccgctgtaca 2340 cctgtgtggc gccgggcgag gtccgggcgt ccaccgaggc caaggcgctc gccgcgcacc 2400 gcgacccgaa gggcttcccg ctcgcggacg cccgccgggt cgccggtctg accggtgtct 2460 accaggtgcc cgcgggcgcc gtgatggaca tcgacctcgg ctcgggcacc gccgtcaccc 2520 accgcacctg gaccccgggc ctctcccgcc gcatcctgcc ggagggcgag gccgtcgcgg 2580 ccgtgcgggc cgcgctggag aaggccgtcg cccagcgggt cacccccggc gacaccccgt 2640 tggtggtgct ctccggcgga atcgactcct ccggggtcgc ggcctgtgcg caccgggcgg 2700 ccggggaact ggacacggtg tccatgggca ccgacacgtc caacgagttc cgcgaggccc 2760 gggcggtcgt cgaccatctg cgcacccggc accgggagat caccatcccg accaccgagc 2820 tgctggcgca gctcccgtac gcggtgtggg cctccgagtc ggtggacccg gacatcatcg 2880 agtacctgct ccccctgaca gcgctctacc gggcgctcga cgggccggag cgccgcatcc 2940 tcaccgggta cggcgcggac atccccctcg ggggcatgca ccgcgaggac cggctgcccg 3000 cgctggacac cgttctcgcg cacgacatgg ccaccttcga cgggctgaac gagatgtccc 3060 cggtgctgtc cacgctggcg gggcactgga ccacccaccc gtactgggac cgggaggtcc 3120 tcgatctgct ggtctcgctg gaggccgggc tcaagcggcg gcacggccgg gacaagtggg 3180 tgctgcgcgc cgcgatggcc gacgccctcc cggcggagac cgtcaaccgg cccaagctgg 3240 gcgtccacga gggctcgggc accacgtcct cgttctcccg gctgctgctg gaccacggtg 3300 tcgccgagga ccgcgtccac gaggcgaagc ggcaggtggt gcgcgagctg ttcgatctca 3360 cggtcggggg cggacggcac ccctccgagg tggacaccga cgatgtggtg cgctccgtgg 3420 ccgaccggac cgcgcggggg gcggcctagt cccgccacgg ggagcccgcc ggacgccgga 3480 cccgcgcggg acccgtaccc ggggccgccc gcggactccg gcgcaccggc acccctgtcc 3540 cccacccgtt gacgaccgtc ggccctcggc cctcgcggcc cctgacgacc gtcgcccgat 3600 tcccaggagg gagctgaaag cgtggagcgc atcgactcgc acgtttcacc ccgctacgca 3660 cagatcccca ccttcatgcg cctgccgcac gatccccagc cccgcggcta tgacgtggtg 3720 gtcatcggag ccccctacga cgggggcacc agctaccgtc ccggcgcccg gttcggcccc 3780 caggccatcc gcagtgagtc gggcctcatc cacggtgtcg gcatcgaccg gggccccggc 3840 acgttcgacc tgatcaactg tgtcgacgcc ggggacatca atctgacgcc gttcgacatg 3900 aacatcgcga tcgacacggc gcagagccat ctgtcgggcc tgctgaaggc caacgccgcc 3960 tttctgatga tcggcggcga ccactcgctg acggtggccg ccctgcgcgc ggtcgcggag 4020 cagcacggcc cgctcgccgt ggtgcacctg gacgcgcact ccgacaccaa cccggccttc 4080 tacgggggcc ggtaccacca cggcaccccc ttccggcacg ggatcgacga gaagctgatc 4140 gacccggcgg cgatggtcca gatcggcatc cggggccaca acccgaagcc ggactcgctc 4200 gactacgccc ggggccacgg cgtccgggtg gtcacggcgg acgagttcgg cgagctgggg 4260 gtgggcggga ccgccgacct catccgcgag aaggtcggcc agcggcccgt gtacgtctcg 4320 gtcgacatcg acgtggtcga ccccgccttc gcccccggta cgggcacgcc cgcgccgggc 4380 gggctcctct cgcgcgaggt gctggcgctg ctgcgctgcg tgggtgacct gaagccggtc 4440 ggcttcgacg tgatggaggt gtcacccctc tacgaccacg gcgggatcac ttcgatcctg 4500 gccacggaga tcggtgcgga actgctctac cagtacgccc gagcccacag aacccagttg 4560 tgaaggagac atcgtgtcat ggcctctccg atagttgact gcaccccgta ccgcgacgag 4620 ctgctcgcgc tcgcctccga gcttcccgag gtgccgcgcg cggacctcca tggcttcctc 4680 gacgaggcga agacgctggc cgcccgtctc ccggaggggc tggccgccgc tctcgacacc 4740 ttcaacgccg tgggcagcga ggacggttat ctgctgctgc gcgggctgcc cgtcgacgac 4800 agcgagctgc ccgagacgcc gacctccacc ccggccccgc tggaccgcaa gcggctggtg 4860 atggaggcca tgctcgcgct ggccggccgc cggctcggtc tgcacacggg gtaccaggag 4920 ctgcgctcgg gcacggtcta ccacgacgtg tacccgtcgc ccggcgcgca ctacctgtcc 4980 tcggagacct ccgagacgct gctggagttc cacacggaga tggcgtacca catcctccag 5040 ccgaactacg tcatgctggc ctgctcccgc gcggaccacg agaaccgggc ggagacgctg 5100 gtcggctcgg tccgcaaggc gctgcccctg ctggacgaga agacccgggc ccgtctcttc 5160 gaccgcaagg tgccctgctg cgtggacgtg gccttccgcg gcggggtcga cgacccgggc 5220 gcgatcgcca acgtcaagcc gctctacggg gacgcgaacg acccgttcct cgggtacgac 5280 cgcgagctgc tggcgccgga ggaccccgcg gacaaggagg ccgtcgccca tctgtcccag 5340 gcgctcgacg atgtgaccgt cggggtgaag ctcgtccccg gtgacgtcct catcatcgac 5400 aacttccgca ccacgcacgc gcggacgccg ttctcgcccc gctgggacgg gaaggaccgc 5460 tggctgcacc gcgtctacat ccgcaccgac cgcaatggac agctctccgg cggcgagcgc 5520 gcgggcgaca ccatctcgtt ctcgccgcgc cgctgagccc ggctccccga ggccctgggc 5580 cccggcgccg gaaccggctc ccggtcctgc cccctcaccc gccgcgcggg tgagggggca 5640 ggcccctttg tgccgggtgc cgtgcgtcct gcgagggtgc cggggcgggg gggacggcgg 5700 aggtgcccgg cggccgggtg ccgtgcgccg cccgtgggtg ctgtacagca ctccgtgtgc 5760 cgtgcgccac cccgtgcata aatttgccac tctatgggaa ataatgcaga gtgcgacggg 5820 tgaggccgtc gccgtgccct ttccgtgaca ggagacgctg acatgtccga cagcacaccg 5880 aagacgcccc ggggattcgt ggtgcacacg gcgccggtgg gcctggccga cgacggccgc 5940 cacgacttca ccgtcctcgc ctccaccgcc ccggccaccg tgagcgccgt cttcacccgc 6000 tcccgcttcg ccgggccgag cgtcgtgctg tgccgggagg cggtggccga cgggcaggcg 6060 cgcggtgtgg tggtgctggc ccgcaacgcg aatgtcgcga ccggcctgga gggcgaggag 6120 aacgcgcgcg aggtgcgcga ggccgtcgcc cgggccctcg ggctgccgga gggcgagatg 6180 ctgatcgcct ccaccggggt gatcggccgg cagtacccga tggagagcat ccgggagcac 6240 ctcaagacgc tggagtggcc cgccggggag ggcggcttcg accgcgcggc ccgcgccatc 6300 atgacgaccg acacccggcc caaggaggtc cgggtcagcg tcggcggggc gaccctcgtg 6360 ggcatcgcca agggcgtcgg catgctggag cccgacatgg cgacgctgct gaccttcttc 6420 gccacggacg cccggctgga cccggccgag caggaccgcc tcttccgccg ggtcatggac 6480 cgcaccttca acgcggtcag catcgacacc gacacctcca ccagcgacac ggcggtgctg 6540 ttcgccaacg gcctggcggg cgaggtcgac gccggggagt tcgaggaggc gctgcacacg 6600 gcggcgctgg ccctggtcaa ggacatcgcg agcgacggcg agggcgcggc caagctgatc 6660 gaggtccagg tcaccggcgc ccgcgacgac gcccaggcca agcgggtcgg caagaccgtc 6720 gtcaactccc cgttggtgaa gaccgccgtg cacggctgcg accccaactg gggccgggtc 6780 gccatggcga tcggcaagtg ctcggacgac accgacatcg accaggagcg ggtgacgatc 6840 cgcttcggcg aggtcgaggt ctatccgccg aaggcccggg gcgaccaggc cgacgacgcg 6900 ctgcgggccg ccgtcgcgga gcatctgcgg ggcgacgagg tggtcatcgg gatcgacctc 6960 gccatcgcgg acggggcctt caccgtctac ggctgcgacc tcaccgaggg ctatgtccgg 7020 ctgaactcgg agtacaccac ctgatccccg gacagggaac gggccgccgc cccgttccct 7080 gtccgctccc gtcccgtgtg gttataccga ccgttccccg gctatgcgca cgggacggag 7140 cggcccccgc cgggccccgc ccggccgcac gatgaggggc gatgcaaggt gacgagggca 7200 ggagggacat ggagaccact cggtcgacga ccgcggacga gggcttcgac gccggggtac 7260 ggggagtggt cgcgccgacc gacgccccgg gcgggacgct gcggctggtc cgcacggacg 7320 acttcgactc gctcgacccc ggcaacacgt actacgccta cacctggaac ttcctccggc 7380 tcatcggccg gacgctggtc accttcgaca ccgcgccggg caaggcgggc cagcggctcg 7440 tgcccgacct cgccgagtcg ctgggcgagt cctccgagga cggccgggtc tggacctacc 7500 ggctgcgcga gggcctgcgc tacgaggacg gcacgccggt cgtctcggcc gacatcaagc 7560 acgccatcgc ccgcagcaac tacggcaccg atgtcctggg cgccggtccg acctacttcc 7620 gccacctcct gggcaccgag tacggcggcc cctggcggga gccggacgcc gacggaccgg 7680 tgacgctgga gaccccggac gagcggacgc tggtcttccg gctgcgggag ccgttcgcgg 7740 ggatggatct gctggcgacc atgccgtcca ccacccccgt gccgcgcgac cgggacaccg 7800 gcgccgagta ccggctgcgg cccgtggcga ccggcccgta ccggatcgtc tcgtacaccc 7860 ggggcgagct ggccgtcctg gagcccaatc cgcactggga ccccgagacc gacccggtgc 7920 gcgtccagcg cgcctcccgg atcgaggtgc acctcggcaa ggacccgcac gaggtggacc 7980 gcatgctgct ggcgggcgag gcccatgtgg acctcgcggg cttcggtgtg cagcccgcgg 8040 cccaggagcg catcctcgcc gagccggagc tgcgcgcgca cgcggacaac ccgctgaccg 8100 gcttcacctg gatctactgc ctgtcgagcc ggatcgcccc gttcgacaat gtgcactgcc 8160 ggcgggccgt gcagttcgcc accgacaaag cggccatgca ggaggcgtac ggcggcgcgg 8220 tgggcggcga catcgcgacc accctgctgc ccccgaccct cgacggctac aagcacttcg 8280 accgctaccc ggtcggcccc gagggcaccg gcgacctgga ggccgcccgc gccgagctga 8340 agctggccgg gatgcccgac ggcttccgca ccaggatcgc cgcccgcaag gaccggctca 8400 aggagtaccg ggccgccgag gcgctggccg ccgggctcgc ccgggtcggc atcgaggcgg 8460 aggtgctgga cttcccgtcg ggcgactact tcgaccgcta cggcggctgc ccggagtatc 8520 tgcgcgagca cgggatcggg atcatcatgt tcggctgggg cgccgacttc cccgacggat 8580 acggcttcct ccagcagatc accgacgggc gcgcgatcaa ggagcgcggc aaccagaaca 8640 tgggcgagct ggacgacccg gagatcaacg cgctgctgga cgagggggcg cagtgcgccg 8700 acccggcgcg gcgcgcggag atctggcacc gcatcgacca gctcacgatg gaccacgcgg 8760 tcatcgttcc gtatctgtac ccgcggtccc tgctctaccg gcacccggac acccgcaacg 8820 ccttcgtcac cggctccttc gggatgtacg actacgtggc gctcggcgcg aagtgagcac 8880 ggggtccggc cccgggaccg tatgtcccgg ggccggaccc cgcccgttcc ccgcccggtc 8940 cggtccggac ccggtcgcgg cccgctcagc cggacatccg ggccccggcc gcgaccccgc 9000 gccggatcgg ccagtggccc tgcgccaggg gccgttccac gctgcggcag gcgagagcgg 9060 cctcgcggaa ctccgcctcg tacagcgcga gctggcgcag gaactgccgg gtcgggccgg 9120 tcaggctggt cccccgcggg ctgcgcagca gcagccgggc gccgagggac tgctccagcc 9180 ggtgaatccg gcgggtgagc gccgactggc tgatcgacag caccgccgcg gcccggttga 9240 tgctgccgtg ccgggccacg gcctggagca gatggagatc gtccacatcc agtttgcggc 9300 cctcggcctg gccgggcacg gagccctggt cgggtcccgc cccgaagcgg cgggcgtccg 9360 cgccggtgcg ctccgcgtac cactgcgccc accagggctc gtccagcagg tcgcggtggt 9420 gttcggcgaa gcgccggagc tggacctcgg cgatcagcgc ggccagccgt cccgccagcg 9480 cccggggcac gatggtgggg tcgacgagca gactcgtggt gcggcgcggg cgctccgcca 9540 gggagcggcg caccagcgag gggtcctgca ccgccgggtg ggtgggcgag ccgagaccta 9600 tcgcgtcccc gcggcgcagg atgccccggg caaccgatgc ccccgtgatg tggagccggg 9660 tgggcgcggt gagcccggcc agctggaaga cacgtgtcac caggatctcc gagccgggtc 9720 ccgtctcgga cacccaggtc tcgtcccgca gatcggcgag cgagacctcc cgccgggcgg 9780 ccagcggatg gtcccggggc aggatcaccc acagcgggtc gtccagcacc tcacaggtgc 9840 gcacggaccg ctccaggctg tgccgggggg actggaggct ccaggtgtag gccgcgtcca 9900 cctggtagcc cgccagttgg gcggcgacct ggtgcggggc ctcgtgccgg accgacagca 9960 gcaggtccag cgaggccgcc gcgtcctcca ccacctcgtc gagcaggggt tccgtggaga 10020 ccagcgacag cacctccggg gcgtccacgg cctcggagcc atggccgaag atatgcgtcc 10080 gcgcggccag gtcgacctgg tggaagaacc gccgcccggc gacgaggatg cgggagcccg 10140 cggtggtcag ccgggccgtg tggcggctgc gcagggtcag cgggaggccg acgatccggt 10200 ccagccggtc gagtctgcgc tccacggtgc cgtgccggac acccgtccgc cgggccactt 10260 ccatgaggtc tccgcagtgt cccaccgcgt ccagtaaaga cagatcgcat cggctgacac 10320 cagcagacgt cggttctgac ccgagagaca atgtcggttc ccttttccgt caaggactgt 10380 accgctgaat tgtccgaagt ggctcttgaa ttgcttcgga atcgatccta ggcagcgccg 10440 ctcttcggat tctcctcgcc gggaagcgga acgcgcccgg ccggatggcg ggcgcgctcc 10500 gggcgccgtc ccgggaacgg gggacggggc acggcacggc cggccacccg gtccgggcgc 10560 gcggcgtgga cctggtcggc ggacgggtgt cagacctggt cggtggggcg tatgaagatc 10620 tcgtggacgg tcgcgtggtg cggcgcggtc acggcgtagc ggaccgcctc cgcgatgtcc 10680 tgggcctgga gcttgcggat ctggctgatc cgctgctcgt acatctcctt ggtggcggtg 10740 tgggtgatgt ggccgcgcag ctccgtgtcg gtggtgcccg gctcgatgac gacgacccgc 10800 accccgcgct cggtgacctc ctggcgcagc gtctcgctga acgcgttcac accgaacttc 10860 gtggcctggt agacggccgc gttgcggacg ttcacccggc ccgcgatcga ggacatctgc 10920 accacggtgc ccttgctgcg cagcagatgg ggaagggccg cccgggtcat gtacatcagg 10980 cccaggagat tggtgtcgat catccgggtc cagtcggtgg tgtcggcgtc ctccaccggg 11040 ccgagcagca tgatcccggc gttgttgacg aggatgtcga ggccgcccag cgcctcgacg 11100 gtggaggcga cggcggcgtc caccccctgc cggtcggcga cgtcgagttc gaggacatgg 11160 accttcgccc cggcggcggt cagctcgtca cccagggcgc gcagcttctc gacccggcgc 11220 gcggcgatgg ccacggcggc gccctcggcg gccagggcgc gggccgtggc ctcgccgatg 11280 cccgagctcg cgcccgtgat gagcgcgact ttcccctgga gtgcggatgg catcatttcc 11340 tccacatggt gctgcgatcg tggtgagcgt atgaagaagg ggtgagacct gccgtgccgg 11400 ggcgggttcc gtacgccgga ccgttgcggt gggcacggcc gaccgggtac ggatggccgc 11460 agttccccgg ggagttcccg gggaatggtg aataccgcgg cgctctccga tggtcttcgg 11520 aggacacccg gggattcacc gggaatcagc ggccggagtt ctccccgtcc acggcagacg 11580 ctatcagcgt cgcattcccc ggtg 11604 14 1656 DNA Streptomyces clavuligerus 14 tcagccggcc gcgaggttgc tgaggaactt cgcggcgacg gggcccgcgt cggcgccgcc 60 cgacccgccg tcctccagca ggaccgacca ggcgatgttc cggtcgccct ggtagccgat 120 catccaggcg tgcgtcttcg gcggcttctc ggtgccgaac tcggcggtac cggtcttggc 180 gtgcggctgt ccgccgaggc cccgcagggc gtcgccggcg ccgtcggtga cggtcgaacg 240 catcatggaa cgcagcgagt cgacgatgcc cggggccatc cggggggcct ggtgcggctt 300 cttgaccgcg tcgggcacca gcacgggctg cttgaactcg ccctgcttga cggtggcggc 360 gatggaggcc atcaccaggg gcgacgcctc gaccctggcc tgtccgatgg tggacgcggc 420 cttgtcgttc tcgctgttgg agacggggac gctgccgtcg aaggtggagg cgccgacgtc 480 ccaggtgccg ccgatgccga aggcttcggc ggcctgcttc aggctggact cggagagctt 540 gctgcgggag ttgacgaaga acgtgttgca ggagtgggcg aagctgtccc ggaaggtcga 600 gcccgcgggc agcgtgaact ggtcctggtt ctcgaagctc tggccgttga catgggcgaa 660 cttcgggcag tcggcccgct cctccgggtt catcccctgc tggagcaggg ccgcggtggt 720 gaccaccttg aaggtggagc cgggcgggta gcggccctcc agcgcgcggt tcatgccgga 780 gggcacgttc gcggcggcca ggatgttgcc ggtggcgggg tcgacggcga cgatcgccgc 840 gttcttcttc gagccctcca gggccgccgc ggcggcggac tggacccgcg ggtcgatggt 900 ggtcttcacc ggcttgccct cggtgtcctt gaggccggtg agcttcttga ccacctggcc 960 ggactcacgg tccaggatca cgaccgagcg cgccgcgccg gagccgccgg tgagctgctt 1020 gtcgtagcgg gactggaggc ccgccgagcc cttgccggtc ctggggtcga ccgcgccgat 1080 gatggaggcg gcctggagga cattgccgtt ggcgtcgagg atgtccgcgc gctcccgcga 1140 cttgagggcg agggtctgcc ccggaaccat ctgcggatgg atcatctcgg tgttgaacgc 1200 gaccttccac tccttgccgc cgccgacgac cttcgcggtg gagtcccagg cgtactcccc 1260 ggccccgggg agggtcattc tgacggtgaa cggtatctcc acctcgccct cggggttctt 1320 ctccccggtc ttggcggtga tctccgtctt cgtcggcttg aggttggtca tgacggattt 1380 gatcagcgac tcggcgttgt ccggggtgtc cgtcagcccg gcggccgtcg gggcgtcgcc 1440 cttctcccag gcgccgagga aggtgtcgaa ctgtccggcc gccgcctcca cctcggggtc 1500 gcccgaatcc ttctcgtcgg caaccaggct ggtgtaaccc caatagccga gccccaccgt 1560 cacggccagc ccggcgacca ccgcggtggc cgcccggcca cgggagcggc gcctgccctg 1620 cggcgggtca tcgccatagt tgtcggaatg cgtcat 1656 15 1722 DNA Streptomyces clavuligerus 15 atgtcccgtg tatcgaccgc ccccagcggc aagcctaccg ccgctcacgc cctcctgtca 60 cggttgcgtg atcacggtgt ggggaaggtg tttggggttg tcggccgaga ggccgcgtcg 120 attctcttcg acgaggtcga ggggatcgac ttcgttctga cccgccacga gttcaccgcg 180 ggtgtcgccg ctgatgtcct cgcgcggatc accggtcgcc cccaggcgtg ctgggccacc 240 ctgggccccg gtatgaccaa cctctccacc ggtatcgcca cgtccgtcct ggaccgctcg 300 ccggtcatcg cgctcgccgc gcagtcggag tcgcacgaca tcttcccgaa cgacacccac 360 cagtgcctgg actcggtggc gatcgtcgcc ccgatgtcca agtacgccgt ggagctccag 420 cggccccacg agatcaccga cctcgtcgac tccgccgtga acgcggccat gaccgagccg 480 gtcgggccct ccttcatctc cctcccggtg gacctgctcg gctcctccga gggcatcgac 540 accaccgtcc ccaacccgcc ggcgaacacc ccggcgaaac cggtcggcgt cgtcgccgac 600 ggctggcaga aggccgccga ccaggccgcc gccctgctcg ccgaggccaa gcacccggtg 660 ctcgtcgtcg gagcggccgc gatccgctcg ggcgccgtcc cggcgatccg cgccctggcc 720 gagcgcctga acatcccggt catcacgacc tacatcgcca agggtgtcct gccggtcggc 780 cacgagctga actacggcgc cgtcaccggc tacatggacg gcatcctcaa cttcccggcg 840 ctccagacca tgttcgcccc ggtggacctc gtcctcaccg tcggctacga ctacgccgag 900 gacctgcgcc cgtccatgtg gcagaagggc atcgagaaga agaccgtccg tatctccccg 960 acggtcaacc cgatcccccg ggtctaccgg cccgacgtcg acgtcgtcac cgacgtcctc 1020 gccttcgtgg agcacttcga gaccgcgacc gcctccttcg gggccaagca gcgccacgac 1080 atcgagccgc tgcgcgcccg gatcgcggag ttcctggccg acccggagac ctacgaggac 1140 ggcatgcgcg tccaccaggt catcgactcc atgaacaccg tcatggagga ggccgccgag 1200 cccggcgagg gcacgatcgt ctccgacatc ggcttcttcc gtcactacgg tgtgctcttc 1260 gcccgcgccg accagccctt cggcttcctc acctcggcgg gctgctccag cttcggctac 1320 ggcatccccg ccgccatcgg cgcccagatg gcccgcccgg accagccgac cttcctcatc 1380 gcgggtgacg gcggcttcca ctccaacagc tccgacctgg agaccatcgc ccggctcaac 1440 ctgccgatcg tgaccgtcgt cgtcaacaac gacaccaacg gcctgatcga gctgtaccag 1500 aacatcggtc accaccgcag ccacgacccg gcggtcaagt tcggcggcgt cgacttcgtc 1560 gcgctcgccg aggccaacgg tgtcgacgcc acccgcgcca ccaaccgcga ggagctgctc 1620 gcggccctgc gcaagggtgc cgagctgggt cgtccgttcc tcatcgaggt cccggtcaac 1680 tacgacttcc agccgggcgg cttcggcgcc ctgagcatct ga 1722 16 1542 DNA Streptomyces clavuligerus 16 atgggggcac cggttcttcc ggctgccttc gggttcctgg cctccgcccg aacgggcggg 60 ggccgggccc ccggcccggt cttcgcgacc cggggcagcc acaccgacat cgacacgccc 120 cagggggagc gctcgctcgc ggcgaccctg gtgcacgccc cctcggtcgc gcccgaccgc 180 gcggtggcgc gctccctcac cggcgcgccc accaccgcgg tgctcgccgg tgagatctac 240 aaccgggacg aactcctctc cgtgctgccc gccggacccg cgccggaggg ggacgcggag 300 ctggtcctgc ggctgctgga acgctatgac ctgcatgcct tccggctggt gaacgggcgc 360 ttcgcgaccg tggtgcggac cggggaccgg gtcctgctcg ccaccgacca cgccggttcg 420 gtgccgctgt acacctgtgt ggcgccgggc gaggtccggg cgtccaccga ggccaaggcg 480 ctcgccgcgc accgcgaccc gaagggcttc ccgctcgcgg acgcccgccg ggtcgccggt 540 ctgaccggtg tctaccaggt gcccgcgggc gccgtgatgg acatcgacct cggctcgggc 600 accgccgtca cccaccgcac ctggaccccg ggcctctccc gccgcatcct gccggagggc 660 gaggccgtcg cggccgtgcg ggccgcgctg gagaaggccg tcgcccagcg ggtcaccccc 720 ggcgacaccc cgttggtggt gctctccggc ggaatcgact cctccggggt cgcggcctgt 780 gcgcaccggg cggccgggga actggacacg gtgtccatgg gcaccgacac gtccaacgag 840 ttccgcgagg cccgggcggt cgtcgaccat ctgcgcaccc ggcaccggga gatcaccatc 900 ccgaccaccg agctgctggc gcagctcccg tacgcggtgt gggcctccga gtcggtggac 960 ccggacatca tcgagtacct gctccccctg acagcgctct accgggcgct cgacgggccg 1020 gagcgccgca tcctcaccgg gtacggcgcg gacatccccc tcgggggcat gcaccgcgag 1080 gaccggctgc ccgcgctgga caccgttctc gcgcacgaca tggccacctt cgacgggctg 1140 aacgagatgt ccccggtgct gtccacgctg gcggggcact ggaccaccca cccgtactgg 1200 gaccgggagg tcctcgatct gctggtctcg ctggaggccg ggctcaagcg gcggcacggc 1260 cgggacaagt gggtgctgcg cgccgcgatg gccgacgccc tcccggcgga gaccgtcaac 1320 cggcccaagc tgggcgtcca cgagggctcg ggcaccacgt cctcgttctc ccggctgctg 1380 ctggaccacg gtgtcgccga ggaccgcgtc cacgaggcga agcggcaggt ggtgcgcgag 1440 ctgttcgatc tcacggtcgg gggcggacgg cacccctccg aggtggacac cgacgatgtg 1500 gtgcgctccg tggccgaccg gaccgcgcgg ggggcggcct ag 1542 17 942 DNA Streptomyces clavuligerus 17 gtggagcgca tcgactcgca cgtttcaccc cgctacgcac agatccccac cttcatgcgc 60 ctgccgcacg atccccagcc ccgcggctat gacgtggtgg tcatcggagc cccctacgac 120 gggggcacca gctaccgtcc cggcgcccgg ttcggccccc aggccatccg cagtgagtcg 180 ggcctcatcc acggtgtcgg catcgaccgg ggccccggca cgttcgacct gatcaactgt 240 gtcgacgccg gggacatcaa tctgacgccg ttcgacatga acatcgcgat cgacacggcg 300 cagagccatc tgtcgggcct gctgaaggcc aacgccgcct ttctgatgat cggcggcgac 360 cactcgctga cggtggccgc cctgcgcgcg gtcgcggagc agcacggccc gctcgccgtg 420 gtgcacctgg acgcgcactc cgacaccaac ccggccttct acgggggccg gtaccaccac 480 ggcaccccct tccggcacgg gatcgacgag aagctgatcg acccggcggc gatggtccag 540 atcggcatcc ggggccacaa cccgaagccg gactcgctcg actacgcccg gggccacggc 600 gtccgggtgg tcacggcgga cgagttcggc gagctggggg tgggcgggac cgccgacctc 660 atccgcgaga aggtcggcca gcggcccgtg tacgtctcgg tcgacatcga cgtggtcgac 720 cccgccttcg cccccggtac gggcacgccc gcgccgggcg ggctcctctc gcgcgaggtg 780 ctggcgctgc tgcgctgcgt gggtgacctg aagccggtcg gcttcgacgt gatggaggtg 840 tcacccctct acgaccacgg cgggatcact tcgatcctgg ccacggagat cggtgcggaa 900 ctgctctacc agtacgcccg agcccacaga acccagttgt ga 942 18 978 DNA Streptomyces clavuligerus 18 atggcctctc cgatagttga ctgcaccccg taccgcgacg agctgctcgc gctcgcctcc 60 gagcttcccg aggtgccgcg cgcggacctc catggcttcc tcgacgaggc gaagacgctg 120 gccgcccgtc tcccggaggg gctggccgcc gctctcgaca ccttcaacgc cgtgggcagc 180 gaggacggtt atctgctgct gcgcgggctg cccgtcgacg acagcgagct gcccgagacg 240 ccgacctcca ccccggcccc gctggaccgc aagcggctgg tgatggaggc catgctcgcg 300 ctggccggcc gccggctcgg tctgcacacg gggtaccagg agctgcgctc gggcacggtc 360 taccacgacg tgtacccgtc gcccggcgcg cactacctgt cctcggagac ctccgagacg 420 ctgctggagt tccacacgga gatggcgtac cacatcctcc agccgaacta cgtcatgctg 480 gcctgctccc gcgcggacca cgagaaccgg gcggagacgc tggtcggctc ggtccgcaag 540 gcgctgcccc tgctggacga gaagacccgg gcccgtctct tcgaccgcaa ggtgccctgc 600 tgcgtggacg tggccttccg cggcggggtc gacgacccgg gcgcgatcgc caacgtcaag 660 ccgctctacg gggacgcgaa cgacccgttc ctcgggtacg accgcgagct gctggcgccg 720 gaggaccccg cggacaagga ggccgtcgcc catctgtccc aggcgctcga cgatgtgacc 780 gtcggggtga agctcgtccc cggtgacgtc ctcatcatcg acaacttccg caccacgcac 840 gcgcggacgc cgttctcgcc ccgctgggac gggaaggacc gctggctgca ccgcgtctac 900 atccgcaccg accgcaatgg acagctctcc ggcggcgagc gcgcgggcga caccatctcg 960 ttctcgccgc gccgctga 978 19 1182 DNA Streptomyces clavuligerus 19 atgtccgaca gcacaccgaa gacgccccgg ggattcgtgg tgcacacggc gccggtgggc 60 ctggccgacg acggccgcga cgacttcacc gtcctcgcct ccaccgcccc ggccaccgtg 120 agcgccgtct tcacccgctc ccgcttcgcc gggccgagcg tcgtgctgtg ccgggaggcg 180 gtggccgacg ggcaggcgcg cggtgtggtg gtgctggccc gcaacgcgaa tgtcgcgacc 240 ggcctggagg gcgaggagaa cgcgcgcgag gtgcgcgagg ccgtcgcccg ggccctcggg 300 ctgccggagg gcgagatgct gatcgcctcc accggggtga tcggccggca gtacccgatg 360 gagagcatcc gggagcacct caagacgctg gagtggcccg ccggggaggg cggcttcgac 420 cgcgcggccc gcgccatcat gacgaccgac acccggccca aggaggtccg ggtcagcgtc 480 ggcggggcga ccctcgtggg catcgccaag ggcgtcggca tgctggagcc cgacatggcg 540 acgctgctga ccttcttcgc cacggacgcc cggctggacc cggccgagca ggaccgcctc 600 ttccgccggg tcatggaccg caccttcaac gcggtcagca tcgacaccga cacctccacc 660 agcgacacgg cggtgctgtt cgccaacggc ctggcgggcg aggtcgacgc cggggagttc 720 gaggaggcgc tgcacacggc ggcgctggcc ctggtcaagg acatcgcgag cgacggcgag 780 ggcgcggcca agctgatcga ggtccaggtc accggcgccc gcgacgacgc ccaggccaag 840 cgggtcggca agaccgtcgt caactccccg ttggtgaaga ccgccgtgca cggctgcgac 900 cccaactggg gccgggtcgc catggcgatc ggcaagtgct cggacgacac cgacatcgac 960 caggagcggg tgacgatccg cttcggcgag gtcgaggtct atccgccgaa ggcccggggc 1020 gaccaggccg acgacgcgct gcgggccgcc gtcgcggagc atctgcgggg cgacgaggtg 1080 gtcatcggga tcgacctcgc catcgcggac ggggccttca ccgtctacgg ctgcgacctc 1140 accgagggct atgtccggct gaactcggag tacaccacct ga 1182 20 1668 DNA Streptomyces clavuligerus 20 atggagacca ctcggtcgac gaccgcggac gagggcttcg acgccggggt acggggagtg 60 gtcgcgccga ccgacgcccc gggcgggacg ctgcggctgg tccgcacgga cgacttcgac 120 tcgctcgacc ccggcaacac gtactacgcc tacacctgga acttcctccg gctcatcggc 180 cggacgctgg tcaccttcga caccgcgccg ggcaaggcgg gccagcggct cgtgcccgac 240 ctcgccgagt cgctgggcga gtcctccgag gacggccggg tctggaccta ccggctgcgc 300 gagggcctgc gctacgagga cggcacgccg gtcgtctcgg ccgacatcaa gcacgccatc 360 gcccgcagca actacggcac cgatgtcctg ggcgccggtc cgacctactt ccgccacctc 420 ctgggcaccg agtacggcgg cccctggcgg gagccggacg ccgacggacc ggtgacgctg 480 gagaccccgg acgagcggac gctggtcttc cggctgcggg agccgttcgc ggggatggat 540 ctgctggcga ccatgccgtc caccaccccc gtgccgcgcg accgggacac cggcgccgag 600 taccggctgc ggcccgtggc gaccggcccg taccggatcg tctcgtacac ccggggcgag 660 ctggccgtcc tggagcccaa tccgcactgg gaccccgaga ccgacccggt gcgcgtccag 720 cgcgcctccc ggatcgaggt gcacctcggc aaggacccgc acgaggtgga ccgcatgctg 780 ctggcgggcg aggcccatgt ggacctcgcg ggcttcggtg tgcagcccgc ggcccaggag 840 cgcatcctcg ccgagccgga gctgcgcgcg cacgcggaca acccgctgac cggcttcacc 900 tggatctact gcctgtcgag ccggatcgcc ccgttcgaca atgtgcactg ccggcgggcc 960 gtgcagttcg ccaccgacaa agcggccatg caggaggcgt acggcggcgc ggtgggcggc 1020 gacatcgcga ccaccctgct gcccccgacc ctcgacggct acaagcactt cgaccgctac 1080 ccggtcggcc ccgagggcac cggcgacctg gaggccgccc gcgccgagct gaagctggcc 1140 gggatgcccg acggcttccg caccaggatc gccgcccgca aggaccggct caaggagtac 1200 cgggccgccg aggcgctggc cgccgggctc gcccgggtcg gcatcgaggc ggaggtgctg 1260 gacttcccgt cgggcgacta cttcgaccgc tacggcggct gcccggagta tctgcgcgag 1320 cacgggatcg ggatcatcat gttcggctgg ggcgccgact tccccgacgg atacggcttc 1380 ctccagcaga tcaccgacgg gcgcgcgatc aaggagcgcg gcaaccagaa catgggcgag 1440 ctggacgacc cggagatcaa cgcgctgctg gacgaggggg cgcagtgcgc cgacccggcg 1500 cggcgcgcgg agatctggca ccgcatcgac cagctcacga tggaccacgc ggtcatcgtt 1560 ccgtatctgt acccgcggtc cctgctctac cggcacccgg acacccgcaa cgccttcgtc 1620 accggctcct tcgggatgta cgactacgtg gcgctcggcg cgaagtga 1668 21 1299 DNA Streptomyces clavuligerus 21 tcagccggac atccgggccc cggccgcgac cccgcgccgg atcggccagt ggccctgcgc 60 caggggccgt tccacgctgc ggcaggcgag agcggcctcg cggaactccg cctcgtacag 120 cgcgagctgg cgcaggaact gccgggtcgg gccggtcagg ctggtccccc gcgggctgcg 180 cagcagcagc cgggcgccga gggactgctc cagccggtga atccggcggg tgagcgccga 240 ctggctgatc gacagcaccg ccgcggcccg gttgatgctg ccgtgccggg ccacggcctg 300 gagcagatgg agatcgtcca catccagttt gcggccctcg gcctggccgg gcacggagcc 360 ctggtcgggt cccgccccga agcggcgggc gtccgcgccg gtgcgctccg cgtaccactg 420 cgcccaccag ggctcgtcca gcaggtcgcg gtggtgttcg gcgaagcgcc ggagctggac 480 ctcggcgatc agcgcggcca gccgtcccgc cagcgcccgg ggcacgatgg tggggtcgac 540 gagcagactc gtggtgcggc gcgggcgctc cgccagggag cggcgcacca gcgaggggtc 600 ctgcaccgcc gggtgggtgg gcgagccgag acctatcgcg tccccgcggc gcaggatgcc 660 ccgggcaacc gatgcccccg tgatgtggag ccgggtgggc gcggtgagcc cggccagctg 720 gaagacacgt gtcaccagga tctccgagcc gggtcccgtc tcggacaccc aggtctcgtc 780 ccgcagatcg gcgagcgaga cctcccgccg ggcggccagc ggatggtccc ggggcaggat 840 cacccacagc gggtcgtcca gcacctcaca ggtgcgcacg gaccgctcca ggctgtgccg 900 gggggactgg aggctccagg tgtaggccgc gtccacctgg tagcccgcca gttgggcggc 960 gacctggtgc ggggcctcgt gccggaccga cagcagcagg tccagcgagg ccgccgcgtc 1020 ctccaccacc tcgtcgagca ggggttccgt ggagaccagc gacagcacct ccggggcgtc 1080 cacggcctcg gagccatggc cgaagatatg cgtccgcgcg gccaggtcga cctggtggaa 1140 gaaccgccgc ccggcgacga ggatgcggga gcccgcggtg gtcagccggg ccgtgtggcg 1200 gctgcgcagg gtcagcggga ggccgacgat ccggtccagc cggtcgagtc tgcgctccac 1260 ggtgccgtgc cggacacccg tccgccgggc cacttccat 1299 22 744 DNA Streptomyces clavuligerus 22 tcagacctgg tcggtggggc gtatgaagat ctcgtggacg gtcgcgtggt gcggcgcggt 60 cacggcgtag cggaccgcct ccgcgatgtc ctgggcctgg agcttgcgga tctggctgat 120 ccgctgctcg tacatctcct tggtggcggt gtgggtgatg tggccgcgca gctccgtgtc 180 ggtggtgccc ggctcgatga cgacgacccg caccccgcgc tcggtgacct cctggcgcag 240 cgtctcgctg aacgcgttca caccgaactt cgtggcctgg tagacggccg cgttgcggac 300 gttcacccgg cccgcgatcg aggacatctg caccacggtg cccttgctgc gcagcagatg 360 gggaagggcc gcccgggtca tgtacatcag gcccaggaga ttggtgtcga tcatccgggt 420 ccagtcggtg gtgtcggcgt cctccaccgg gccgagcagc atgatcccgg cgttgttgac 480 gaggatgtcg aggccgccca gcgcctcgac ggtggaggcg acggcggcgt ccaccccctg 540 ccggtcggcg acgtcgagtt cgaggacatg gaccttcgcc ccggcggcgg tcagctcgtc 600 acccagggcg cgcagcttct cgacccggcg cgcggcgatg gccacggcgg cgccctcggc 660 ggccagggcg cgggccgtgg cctcgccgat gcccgagctc gcgcccgtga tgagcgcgac 720 tttcccctgg agtgcggatg gcat 744 23 1227 DNA Streptomyces clavuligerus 23 atgatgaacg aggcagcgcc tcagtccgac caggtggcac cggcgtatcc gatgcaccgg 60 gtctgcccgg tcgacccgcc gccgcaactg gccgggctgc ggtcccagaa ggccgcgagc 120 cgggtgacgc tgtgggacgg cagccaggtg tggctggtga cctcgcacgc cggggcccgg 180 gccgtcctgg gcgaccgccg cttcaccgcg gtgacgagcg cgcccggctt cccgatgctg 240 acccgcacct cccaactggt gcgcgccaac ccggagtcgg cgtcgttcat ccgcatggac 300 gacccgcagc actcccggct gcgctcgatg ctcacccggg acttcctggc ccgccgcgcc 360 gaggcgctgc gccccgcggt gcgggagctg ctggacgaga tcctgggcgg gctggtgaag 420 ggggagcggc cggtcgacct ggtcgccgga ctgacgatcc cggtgccctc gcgggtcatc 480 accctgctct tcggcgccgg tgacgaccgc cgggagttca tcgaggaccg cagcgcggtc 540 ctcatcgacc gcggctacac cccggagcag gtcgccaagg cccgggacga actcgacggc 600 tatctgcggg agctggtcga ggagcggatc gagaacccgg gcaccgacct gatcagccgg 660 ctcgtcatcg accaggtgcg gccggggcat ctgcgggtcg aggagatggt cccgatgtgc 720 cggctgctgc tggtggccgg tcacggcacc accaccagcc aggcgagcct gagcctgctc 780 agcctgctca ccgacccgga gctggccggg cgcctcaccg aggacccggc cctgctgccc 840 aaggcggtcg aggagctgct gcgcttccac tccatcgtgc agaacgggct ggcccgtgcc 900 gcggtggagg acgtccagct cgacgatgtg ctcatccggg cgggcgaggg cgtggtgctg 960 tcgctgtcgg cgggcaaccg ggacgagacg gtcttccccg acccggaccg ggtggacgtg 1020 gaccgcgacg cccgccgcca tctcgccttc ggccacggca tgcaccagtg cctgggccag 1080 tggctggccc gggtggagct ggaggagatc ctcgccgcgg tgctgcgctg gatgcccggt 1140 gcccggctcg cggtgccctt cgaggagctg gacttccgtc atgaggtgtc cagttacggc 1200 ctcggcgccc tcccggtgac ctggtga 1227 24 24 DNA Artificial Sequence primer 24 taygcncara thccnacntt yatg 24 25 24 DNA Artificial Sequence primer 25 tacgcscaga tcccsacstt catg 24 

We claim:
 1. An isolated DNA molecule consisting of the nucleotide sequence selected from the group consisting of SEQ ID NO:15, 16, 19, 20, 21, and
 22. 2. The isolated DNA molecule of claim 1 consisting of the nucleotide sequence of SEQ ID NO:15.
 3. The isolated DNA molecule of claim 1 consisting of the nucleotide sequence of SEQ ID NO:16.
 4. The isolated DNA molecule of claim 1 consisting of the nucleotide sequence of SEQ ID NO:19.
 5. The isolated DNA molecule of claim 1 consisting of the nucleotide sequence of SEQ ID NO:20.
 6. The isolated DNA molecule of claim 1 consisting of the nucleotide sequence of SEQ ID NO:21.
 7. The isolated DNA molecule of claim 1 consisting of the nucleotide sequence of SEQ ID NO:22.
 8. An isolated DNA molecule consisting of a nucleotide sequence encoding the amino acid sequence selected from the group consisting of SEQ ID NO:4, 5, 8, 9, 10 and
 11. 9. The isolated DNA molecule of claim 8 consisting of the nucleotide sequence of SEQ ID NO:4.
 10. The isolated DNA molecule of claim 8 consisting of the nucleotide sequence of SEQ ID NO:5.
 11. The isolated DNA molecule of claim 8 consisting of the nucleotide sequence of SEQ ID NO:8.
 12. The isolated DNA molecule of claim 8 consisting of the nucleotide sequence of SEQ ID NO:9.
 13. The isolated DNA molecule of claim 8 consisting of the nucleotide sequence of SEQ ID NO:10.
 14. The isolated DNA molecule of claim 8 consisting of the nucleotide sequence of SEQ ID NO:11.
 15. The isolated DNA molecule of claim 1 consisting of the nucleotide sequence of SEQ ID NO:15 and 16, and excluding other reading frames.
 16. The isolated DNA molecule of claim 1 consisting of the nucleotide sequence of SEQ ID NO:19, 20, 21 and 22 and excluding other reading frames.
 17. A recombinant vector comprising at least one of the isolated DNA molecules of claim
 1. 18. A recombinant vector comprising at least one of the isolated DNA molecules of claim
 8. 19. A host transformed with a recombinant vector in accordance with claim
 17. 20. A host in accordance with claim 19 which is a Streptomycete.
 21. A host in accordance with claim 20 which is S. lividans.
 22. A host transformed with a recombinant vector in accordance with claim
 18. 23. A host in accordance with claim 22 which is a Streptomycete.
 24. A host in accordance with claim 23 which is S. lividans.
 25. A process for producing clavulanic acid in a non-clavulanate-producing host comprising transforming the host with at least one of the DNA molecules of claim 1, and culturing the host under suitable conditions to produce clavulanic acid.
 26. A process for producing clavulanic acid in accordance with claim 25 wherein the host is S. lividans.
 27. A process for producing clavulanic acid in a non-clavulanate-producing host comprising transforming the host with at least one of the DNA molecules of claim 8, and culturing the host under suitable conditions to produce clavulanic acid.
 28. A process for producing clavulanic acid in accordance with claim 27 wherein the host is S. lividans.
 29. A process for enhancing clavulanic acid production in a clavulanate-producing host comprising transforming the host with a DNA molecule comprising a nucleotide sequence in accordance with claim
 1. 30. A process for enhancing clavulanic acid production in a clavulanate-producing host comprising transforming the host with a DNA molecule comprising a nucleotide sequence in accordance with claim
 8. 31. An isolated DNA molecule consisting of the nucleotide sequence of nucleotides 2257-5522 of SEQ ID NO:1.
 32. The isolated DNA molecule of claim 31 consisting of the nucleotide sequence of nucleotides1765-6594 of SEQ ID NO:1.
 33. The isolated DNA molecule of claim 34 consisting of the nucleotide sequence of nucleotides 7630-13809 of SEQ ID NO:1.
 34. An isolated DNA molecule consisting of the nucleotide sequence of nucleotides 7936-13,406 of SEQ ID NO:1. 